Explore the full ebook collection and download it now at textbookfull.

com

Brain Development Methods and Protocols Simon G.

Sprecher

https://textbookfull.com/product/brain-development-methods-
and-protocols-simon-g-sprecher/

OR CLICK HERE

DOWLOAD EBOOK

Browse and Get More Ebook Downloads Instantly at https://textbookfull.com

Click here to visit textbookfull.com and download textbook now
 Your digital treasures (PDF, ePub, MOBI) await
Download instantly and pick your perfect format...

Read anywhere, anytime, on any device!

Microbiome Analysis Methods and Protocols Robert G. Beiko

https://textbookfull.com/product/microbiome-analysis-methods-and-
protocols-robert-g-beiko/

textbookfull.com

Yeast Systems Biology Methods and Protocols Stephen G.

Oliver

https://textbookfull.com/product/yeast-systems-biology-methods-and-
protocols-stephen-g-oliver/

textbookfull.com

T Cell Development Methods and Protocols 1st Edition Rémy

Bosselut

https://textbookfull.com/product/t-cell-development-methods-and-
protocols-1st-edition-remy-bosselut/

textbookfull.com

Targeting Enzymes for Pharmaceutical Development Methods

and Protocols Nikolaos E. Labrou

https://textbookfull.com/product/targeting-enzymes-for-pharmaceutical-
development-methods-and-protocols-nikolaos-e-labrou/

textbookfull.com
Zebrafish Methods and Protocols Koichi Kawakami

https://textbookfull.com/product/zebrafish-methods-and-protocols-
koichi-kawakami/

textbookfull.com

SNAREs: Methods and Protocols Rutilio Fratti

https://textbookfull.com/product/snares-methods-and-protocols-rutilio-
fratti/

textbookfull.com

Epitranscriptomics: Methods and Protocols Narendra

Wajapeyee

https://textbookfull.com/product/epitranscriptomics-methods-and-
protocols-narendra-wajapeyee/

textbookfull.com

Phytoplasmas: Methods and Protocols Rita Musetti

https://textbookfull.com/product/phytoplasmas-methods-and-protocols-
rita-musetti/

textbookfull.com

Metalloproteins: Methods and Protocols Yilin Hu

https://textbookfull.com/product/metalloproteins-methods-and-
protocols-yilin-hu/

textbookfull.com
Methods in
Molecular Biology 2047

Simon G. Sprecher Editor

Brain
Development
Methods and Protocols
Second Edition
Methods in M o l e c u l a r B i o lo g y

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in
all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-
by-step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of theMethods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Brain Development

Methods and Protocols

Second Edition

Edited by

Simon G. Sprecher
Department of Biology, University of Fribourg, Fribourg, Switzerland
Editor
Simon G. Sprecher
Department of Biology
University of Fribourg
Fribourg, Switzerland

ISSN 1064-3745     ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-9731-2    ISBN 978-1-4939-9732-9 (eBook)
https://doi.org/10.1007/978-1-4939-9732-9

© Springer Science+Business Media, LLC, part of Springer Nature 2020

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed
to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface

How the brain works remains one of the largest enigmatic questions in modern biology.
Particularly, its cellular diversity, connectivity among neurons, formation of neuronal net-
works, use of distinct neurotransmitter systems, and underlying function for behavior raise
the question of how this highly interconnected organ develops. It is therefore not surpris-
ing that the intersection between developmental biology and neuroscience provides an
exceptional field to address and investigate impacting biological questions. Complementing
findings of an array of distinct animal model systems provide the basis of brain development
research. Our current understanding is based on widely used genetic model systems, includ-
ing the fruit fly, zebra fish, chicken, and mouse. These animal models are particularly
impacting since they allow elaborate genetic manipulations including transgenic expression
systems and conditional knockout or knockdown of developmental genes. However, the
advent of genome editing techniques makes it possible to extend investigations towards
other animals that may thus be used to answer specific questions of molecular neuroscience.
These noncanonical experimental systems further substantiate general principles and mech-
anisms in brain development. Questions that can be investigated often depend on the
methodological accessibility. Therefore, progress and developments in constantly improv-
ing laboratory technologies provide an essential ground for the advancement in the field.
This book aims to provide a comprehensive overview and introduction to widely used
leading-edge techniques on a representative range of animals. A particular focus lies on
recent technical advances in molecular genetics.

Fribourg, Switzerland Simon G. Sprecher

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Part I Invertebrate Models

1 Combining BrdU-Labeling to Detection of Neuronal Markers

to Monitor Adult Neurogenesis in Hydra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   3
Wanda Buzgariu, Marie-Laure Curchod, Chrystelle Perruchoud,
and Brigitte Galliot
2 Reverse Genetic Approaches to Investigate the Neurobiology
of the Cnidarian Sea Anemone Nematostella vectensis . . . . . . . . . . . . . . . . . . . . . . .  25
Jamie A. Havrilak and Michael J. Layden
3 Generating Transgenic Reporter Lines for Studying Nervous System
Development in the Cnidarian Nematostella vectensis . . . . . . . . . . . . . . . . . . . . . . .  45
Fabian Rentzsch, Eduard Renfer, and Ulrich Technau
4 Immunostaining and In Situ Hybridization of the Developing
Acoel Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  59
Elena Perea-Atienza, Brenda Gavilán, Simon G. Sprecher,
and Pedro Martinez
5 Immunostaining of the Embryonic and Larval Drosophila Brain . . . . . . . . . . . . . . .  81
Frank Hirth and Danielle C. Diaper
6 Nonfluorescent RNA In Situ Hybridization Combined with Antibody
Staining to Visualize Multiple Gene Expression Patterns in the Embryonic
Brain of Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  97
David Jussen and Rolf Urbach
7 Analysis of Complete Neuroblast Cell Lineages in the Drosophila
Embryonic Brain via DiI Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Karoline F. Kraft and Rolf Urbach
8 Flybow to Dissect Circuit Assembly in the Drosophila Brain: An Update . . . . . . . . . 137
Emma L. Powell and Iris Salecker
9 Live Cell Imaging of Neural Stem Cells in the Drosophila Larval Brain . . . . . . . . . 153
Karolina Miszczak and Boris Egger
10 CRISPR/Cas9 Genome Editing to Study Nervous System
Development in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Cornelia Fritsch and Simon G. Sprecher
11 The Red Flour Beetle as Model for Comparative Neural Development:
Genome Editing to Mark Neural Cells in Tribolium Brain Development . . . . . . . . 191
Max S. Farnworth, Kolja N. Eckermann, Hassan M. M. Ahmed,
Dominik S. Mühlen, Bicheng He, and Gregor Bucher

vii
viii Contents

12 A Protocol for Double Fluorescent In Situ Hybridization

and Immunohistochemistry for the Study of Embryonic Brain Development
in Tribolium castaneum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Marita Buescher, Georg Oberhofer, Natalia Carolina Garcia-Perez,
and Gregor Bucher
13 Immunohistochemistry and Fluorescent Whole Mount RNA In Situ
Hybridization in Larval and Adult Brains of Tribolium . . . . . . . . . . . . . . . . . . . . . . 233
Vera S. Hunnekuhl, Janna Siemanowski, Max S. Farnworth, Bicheng He,
and Gregor Bucher
14 X-Ray Microscopy of the Larval Crustacean Brain . . . . . . . . . . . . . . . . . . . . . . . . . 253
Jakob Krieger and Franziska Spitzner
15 Immunolocalization of Neurotransmitters and Neuromodulators
in the Developing Crayfish Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Steffen Harzsch and Caroline Viertel
16 Immunostainings in Nervous System Development
of the Nematode C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Janet S. Duerr
17 Methods in Brain Development of Molluscs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Andreas Wanninger and Tim Wollesen
18 A Simple Method to Identify Ascidian Brain Lineage Cells at Neural Plate
Stages Following In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Clare Hudson
19 Spawning Induction and Embryo Micromanipulation Protocols
in the Amphioxus Branchiostoma lanceolatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Yann Le Petillon, Stéphanie Bertrand, and Héctor Escrivà

Part II Vertebrate Models

20 In Situ Hybridization and Immunostaining of Xenopus Brain . . . . . . . . . . . . . . . . . 363

Kai-li Liu, Xiu-mei Wang, Zi-long Li, Ying Liu, and Rong-qiao He
21 Morpholino Studies in Xenopus Brain Development . . . . . . . . . . . . . . . . . . . . . . . . 377
Jennifer E. Bestman and Hollis T. Cline
22 Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced
Tyramide Signal Amplification and Its Combination with Immunofluorescent
Protein Visualization in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Gilbert Lauter, Iris Söll, and Giselbert Hauptmann
23 Live Morphometric Classification of Sensory Neurons in Larval Zebrafish . . . . . . . 411
Gema Valera and Hernán López-Schier
24 Immunohistochemistry and In Situ Hybridization
in the Developing Chicken Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Richard P. Tucker, Tatsuto Ishimaru, and Qizhi Gong
25 Gene Silencing in Chicken Brain Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Georgia Tsapara, Irwin Andermatt, and Esther T. Stoeckli
 Contents ix

26 Transplantation of Neural Tissue: Quail–Chick Chimeras . . . . . . . . . . . . . . . . . . . . 457

Andrea Streit and Claudio D. Stern
27 Immunohistochemistry and RNA In Situ Hybridization
in Mouse Brain Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
Jinling Liu and Aimin Liu
28 The Cre/Lox System to Assess the Development of the Mouse Brain . . . . . . . . . . . 491
Claudius F. Kratochwil and Filippo M. Rijli
29 In Utero Electroporation to Study Mouse Brain Development . . . . . . . . . . . . . . . . 513
Emilie Pacary and François Guillemot

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
Contributors

Hassan M. M. Ahmed • Department of Developmental Biology, Johann-Friedrich-­

Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Department of Crop Protection, Faculty of Agriculture, University of Khartoum,
Khartoum-North, Khartoum, Sudan
Irwin Andermatt • Neuroscience Center Zurich, Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland
Stéphanie Bertrand • Sorbonne Université, CNRS, Biologie Intégrative des Organismes
Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France
Jennifer E. Bestman • Biology Department, William and Mary, Williamsburg, VA, USA
Gregor Bucher • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Department of Evolutionary Developmental Genetics, Georg-August-University
Göttingen, Göttingen, Germany
Marita Buescher • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany
Wanda Buzgariu • Department of Genetics and Evolution, iGE3, Faculty of Sciences,
University of Geneva, Geneva, Switzerland
Hollis T. Cline • The Dorris Neuroscience Center, The Scripps Research Institute, La
Jolla, CA, USA
Marie-Laure Curchod • Department of Genetics and Evolution, iGE3, Faculty of
Sciences, University of Geneva, Geneva, Switzerland
Danielle C. Diaper • Department of Basic and Clinical Neuroscience, Maurice Wohl
Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience,
King’s College London, London, UK
Janet S. Duerr • Department of Biological Sciences, Ohio University, Athens, OH, USA
Kolja N. Eckermann • Göttingen Graduate Center for Molecular Biosciences,
Neurosciences and Biophysics, Göttingen, Germany; Department of Developmental Biology
Johann-Friedrich-Blumenbach Institute, GZMB, University of Göttingen, Göttingen,
Germany
Boris Egger • Department of Biology, University of Fribourg, Fribourg, Switzerland
Héctor Escrivà • Sorbonne Université, CNRS, Biologie Intégrative des Organismes
Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France
Max S. Farnworth • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Göttingen Graduate Center for Molecular Biosciences, Neurosciences and Biophysics,
Göttingen, Germany; Department of Evolutionary, Developmental Genetics, Georg-
August-University Göttingen, Göttingen, Germany
Cornelia Fritsch • Department of Biology, University of Fribourg, Fribourg, Switzerland
Brigitte Galliot • Department of Genetics and Evolution, iGE3, Faculty of Sciences,
University of Geneva, Geneva, Switzerland

xi
xii Contributors

Natalia Carolina Garcia-Perez • Department of Evolutionary Developmental Genetics,

Johann-Friedrich-Blumenbach Institute, GZMB, University of Göttingen, Göttingen,
Germany
Brenda Gavilán • Departament de Genètica, Universitat de Barcelona, Barcelona, Spain
Qizhi Gong • Department of Cell Biology and Human Anatomy, University of
California, Davis, Davis, CA, USA
François Guillemot • The Francis Crick Institute, London, UK
Steffen Harzsch • Department of Cytology and Evolutionary Biology, Zoological Institute
and Museum, University of Greifswald, Greifswald, Germany
Giselbert Hauptmann • Department of Molecular Biosciences, The Wenner-Gren
Institute, MBW, Stockholm University, Stockholm, Sweden
Jamie A. Havrilak • Department of Biological Sciences, Lehigh University, Bethlehem, PA,
USA
Bicheng He • Department of Evolutionary Developmental Genetics,
Johann-Friedrich-­Blumenbach Institute, GZMB, University of Göttingen,
Göttingen, Germany; Department of Evolutionary Developmental Genetics,
Georg-August-University Göttingen, Göttingen, Germany
Rong-qiao He • State Key Laboratory of Brain and Cognitive Science, Institute of
Biophysics, Chinese Academy of Sciences, Beijing, China
Frank Hirth • Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical
Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's
College London, London, UK
Clare Hudson • Laboratoire de Biologie du Développement de Villefranche-sur-mer
(LBDV), Sorbonne Université, CNRS, Villefranche-sur-mer, France
Vera S. Hunnekuhl • Department of Evolutionary Developmental Genetics, ,
Georg-August-University Göttingen, Göttingen, Germany
Tatsuto Ishimaru • Department of Cell Biology and Human Anatomy,
University of California, Davis, CA, USA
David Jussen • Institute of Genetics, University of Mainz, Mainz, Germany
Karoline F. Kraft • Institute of Genetics, University of Mainz, Mainz, Germany
Claudius F. Kratochwil • Department of Biology, Zoology and Evolutionary Biology,
University of Konstanz, Konstanz, Germany
Jakob Krieger • Department of Cytology and Evolutionary Biology, Zoological Institute
and Museum, University of Greifswald, Greifswald, Germany
Gilbert Lauter • Department of Biosciences and Nutrition, Neo, Karolinska Institutet,
Huddinge, Sweden
Michael J. Layden • Department of Biological Sciences, Lehigh University, Bethlehem, PA,
USA
Yann Le Petillon • Sorbonne Université, CNRS, Biologie Intégrative des Organismes
Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, Banyuls-sur-Mer, France
Zi-long Li • State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, Beijing, China
Aimin Liu • Department of Biology, Center for Cellular Dynamics, Eberly College of
Science, Huck Institute of Life Sciences, The Penn State University, Pennsylvania, PA,
USA
 Contributors xiii

Jinling Liu • Department of Biology, Center for Cellular Dynamics, Eberly College of
Science, Huck Institute of Life Sciences, The Penn State University, Pennsylvania, PA,
USA
Kai-li Liu • State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, Beijing, China
Ying Liu • State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, Beijing, China
Hernán López-Schier • Sensory Biology and Organogenesis, Helmholtz Zentrum Munich,
Munich, Germany
Pedro Martinez • Departament de Genètica, Universitat de Barcelona, Barcelona,
Spain; Institut Català de Recerca i Estudis Avancats (ICREA), Barcelona, Spain
Karolina Miszczak • Department of Biology, University of Fribourg, Fribourg,
Switzerland
Dominik S. Mühlen • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Göttingen Graduate Center for Molecular Biosciences, Neurosciences and Biophysics,
Göttingen, Germany
Georg Oberhofer • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany
Emilie Pacary • INSERM U1215, Neurocentre Magendie, Bordeaux, France; Université
de Bordeaux, Bordeaux, France
Elena Perea-Atienza • Departament de Genètica, Universitat de Barcelona, Barcelona,
Spain
Chrystelle Perruchoud • Department of Genetics and Evolution, iGE3, Faculty of
Sciences, University of Geneva, Geneva, Switzerland
Emma L. Powell • Visual Circuit Assembly Laboratory, The Francis Crick Institute,
London, UK
Eduard Renfer • Department for Molecular Evolution and Development, Faculty of Life
Sciences, Center of Organismal Systems Biology, University of Vienna, Vienna, Austria
Fabian Rentzsch • Sars International Centre for Marine Molecular Biology, University of
Bergen, Bergen, Norway; Department of Biological Sciences, University of Bergen, Bergen,
Norway
Filippo M. Rijli • Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
Iris Salecker • Visual Circuit Assembly Laboratory, The Francis Crick Institute, London,
UK
Janna Siemanowski • Department of Evolutionary Developmental Genetics,
Georg-August-University Göttingen, Göttingen, Germany
Iris Söll • Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm
University, Stockholm, Sweden
Franziska Spitzner • Department of Cytology and Evolutionary Biology, Zoological
Institute and Museum, University of Greifswald, Greifswald, Germany
Simon G. Sprecher • Department of Biology, University of Fribourg, Fribourg,
Switzerland
Claudio D. Stern • Department of Cell and Developmental Biology, University College
London, London, UK
Esther T. Stoeckli • Neuroscience Center Zurich, Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland
xiv Contributors

Andrea Streit • Faculty of Dental, Oral and Craniofacial Sciences, Centre for
Craniofacial and Regenerative Biology, King’s College London, London, UK
Ulrich Technau • Department for Molecular Evolution and Development, Faculty of
Life Sciences, Center of Organismal Systems Biology, University of Vienna, Vienna,
Austria
Georgia Tsapara • Neuroscience Center Zurich, Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland
Richard P. Tucker • Department of Cell Biology and Human Anatomy, University of
California, Davis, CA, USA
Rolf Urbach • Institute of Genetics, University of Mainz, Mainz, Germany
Gema Valera • Sensory Biology and Organogenesis, Helmholtz Zentrum Munich, Munich,
Germany
Caroline Viertel • Department of Cytology and Evolutionary Biology, Zoological
Institute and Museum, University of Greifswald, Greifswald, Germany
Xiu-mei Wang • State Key Laboratory of Brain and Cognitive Science, Institute of
Biophysics, Chinese Academy of Sciences, Beijing, China
Andreas Wanninger • Department of Integrative Zoology, Faculty of Life Sciences,
University of Vienna, Vienna, Austria
Tim Wollesen • European Molecular Biology Laboratory, Heidelberg, Germany
 Part I

Invertebrate Models
 Chapter 1

Combining BrdU-Labeling to Detection of Neuronal

Markers to Monitor Adult Neurogenesis in Hydra
Wanda Buzgariu, Marie-Laure Curchod, Chrystelle Perruchoud,
and Brigitte Galliot

Abstract
The nervous system is produced and maintained in adult Hydra through the continuous production of
nerve cells and mechanosensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in
intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a
complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie
these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here
we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal
markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as
RFamide or Hym-355. This method can be performed on whole-mount animals as well as on macerated
tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling
can be combined with the analysis of gene expression by whole-mount in situ hybridization. This co-­
immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogen-
esis. Upon continuous BrdU labeling, the repeated measurements of BrdU-labeling indexes in specific
cellular populations provide a precise monitoring of nematogenesis as well as neurogenesis, in homeostatic
or developmental conditions.

Key words Hydra nervous system, Interstitial stem cells, Neurogenesis, Nematogenesis, In situ
hybridization, Immunofluorescence, Hydroxyurea, BrdU, prdl-a, Hym-355, RFamide

1 Introduction

1.1 Anatomy The freshwater hydrozoan Hydra belongs to Cnidaria, an early-­

of the Nervous branched eumetazoan phylum, sister group to bilaterians. Hydra is
System, Neurogenesis, an attractive model for biologists not only for its outstanding
and Nematogenesis regenerative properties but also for its highly dynamic neurogene-
in Hydra sis. Indeed the nervous system is continuously renewed with nerve
precursors produced in the body column, then displaced towards
the extremities where they terminally differentiate to replace the
nerve cells that die [1] (Fig. 1a). In addition, animals bisected at
any level along the body column replace the missing part in few

Simon G. Sprecher (ed.), Brain Development: Methods and Protocols, Methods in Molecular Biology, vol. 2047,
https://doi.org/10.1007/978-1-4939-9732-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

3
4 Wanda Buzgariu et al.

A INTACT HYDRA POLYP C

apical differentiated
nerve cells
interstitial stem cells
sensory neurons

neurogenesis
nerve cell precursors

migration
interstitial stem cells 4x
nerve cell precursors

basal differentiated 8x
nerve cells
sensory-motor bipolar neurons

B HEAD-REGENERATING HYDRA
16

a.p a.p

nematoblasts

4 hpa 24 hpa 36 hpa

a.p=amputation plane ganglia neurons
nematocytes

Fig. 1 Adult neurogenesis in Hydra and cell types that form its nervous system. (a, b) Neurogenesis in adult
intact (a) and in head-regenerating (b) Hydra polyp (modified from ref. 2). Only nerve cells are shown, denser
in the apical (top) and basal (bottom) regions than in the body column where neurogenesis takes place. The
interstitial stem cells (ISCs) provide precursors for all nerve cells and nematocytes, which migrate towards the
extremities [3]. Terminal differentiation of nerve cells predominantly takes place at the extremities. In head-­
regenerating Hydra (b) interstitial cells and derivatives are first eliminated upon injury-induced cell death after
mid-gastric bisection [4] and de novo neurogenesis takes place at the tip where the apical nervous system get
built within 2 days, with precursors detected after 24 h [5, 6]. (c) ISCs divide every 24–30 h and frequently
appear as pairs (top-left panel). Nematogenesis starts with a series of synchronous syncytial divisions that
lead to the formation of nematoblast clusters that contain 4×, 8×, 16× or even 32× cells. At any stage, a
cluster can stop proliferating to enter differentiation, i.e. each nematoblast forms a venom capsule named
nematocyst (arrows) and a cnidocil (not visible), both fully mature in nematocytes [7, 8]. Note the moon-shaped
nucleus in nematocytes, compressed by the nematocyst. Hydra differentiates different classes of neurons:
sensory, sensory-motor bipolar, and multipolar ganglia neurons (right panels). Cells obtained after tissue mac-
eration [9], were immunodetected with an anti α-tubulin antibody followed by an anti-mouse secondary anti-
body coupled with Alexa-488 (green). The nuclei were counterstained with DAPI (light blue). Pictures were
taken on a SP8 Leica confocal microscope and optimized on Photoshop with channelmixer to lighten the dark
blue color and M-curves to increase the contrast. Scale bar: 10 μm

days, e.g. a new head equipped with a complete nervous system

(Fig. 1b). The Hydra nervous system is made of nerve cells and
mechanosensory cells (named nematocytes or cnidocytes) that
form a nerve net much denser at the apical and basal extremities of
the animal. There it controls a series of more or less complex
behaviors such as peristaltic movements, walking, prey capture,
food ingestion, and reaction to light.
Cnidarian neurons are equipped with neurites but no axons,
therefore not polarized and often named “nerve cells.” Every
nerve cell is able to function as sensory, sensory-motor, or inter-
 Methods to Monitor Adult Neurogenesis in Hydra 5

neuron. Nevertheless, nerve cells show diverse anatomies, sensory,

bipolar, or multipolar as ganglia neurons (Fig. 1c), and the neuro-
nal phenotype of a given cell can change according to its position
along the body axis [10]. At the base of the apex, ganglia neurons
can form a nerve ring that allows coordinated behaviors [11].
Although cnidarian nervous systems are highly peptidergic with
peptide-gated ion channels as receptors [12, 13], they share with
bilaterians all the basic properties of synaptic conduction and
chemical neurotransmission [14, 15], even though the neuromus-
cular junction might have evolved independently in cnidarians and
bilaterians [16]. Still, the transcriptional factors that control neu-
rogenesis are largely evolutionarily conserved [2, 5, 9, 17–21] and
cnidarian nervous systems might represent the first evolutionary
attempt of centralized nervous system in eumetazoans [11].
The two myoepithelial layers of the body wall, the epidermis
and the gastrodermis, are made of cells that derive from three not
interchangeable stem cell populations: the two unipotent ectoder-
mal and endodermal epithelial stem cells (ESCs) and the multipo-
tent interstitial stem cells (ISCs). ISCs, predominantly located in
the central body column, give rise to a variety of cell types, includ-
ing the two cell lineages that build up the nervous system (Figs. 1a
and 2): the rare nerve cells and the abundant nematocytes, which
represent 3% and 35% of the total cell number, respectively [41,
42]. The nerve cells directly differentiate from migrating precur-
sors and then get displaced towards the extremities where they
form a dense diffuse nerve net [6, 42–44]. In intact animals
(homeostasis), neurogenesis is a slow process, leading to the
replacement of the nerve cells that get sloughed off at the extremi-
ties, while after bisection a faster de novo neurogenesis process
takes place in the regenerating structure (Fig. 3a).
In contrast to neurogenesis, nematogenesis is a multiple step
process where interstitial progenitors synchronously divide up to
five times, providing clusters that contain 4, 8, 16, or 32 nemato-
blasts (Figs. 1b and 2) [7]. Cells from a given cluster can exit the
cell cycle at any time from the 4-cell stage to differentiate as nema-
tocytes equipped with a sensory cnidocil and a vacuole named cni-
docyst that contains a paralyzing venom, the latter structure being
the hallmark of cnidarians [8]. The turnover of nematocytes is
highly dynamic as once the venom capsule is discharged, the
­nematocyte is eliminated and replaced. Interestingly, ISCs cycle
three to four times faster than ESCs and can thus be easily elimi-
nated upon pulse treatments of antimitotic drugs [45–47].
Elimination of the cycling interstitial cells leads to the loss of the
nervous system, after several weeks in intact animals, within few
days in regenerating ones, as such animals regenerate a missing
part that lacks a nervous system (Figs. 3b and 4). Nerve-free ani-
mals actually maintain their developmental properties if they are
6 Wanda Buzgariu et al.

Fig. 2 Molecular markers of neurogenesis and nematogenesis in Hydra. A partial list of genes expressed during
neurogenesis (upper) and/or nematogenesis (lower). Gene products detected with antibodies are written plain.
ISC interstitial stem cells, pc precursor cell, nb nematoblasts. The spatial and cell-type expression pattern of
genes presumably involved in neurogenesis and neurotransmission (103 and 156, respectively) was identified
by RNAseq transcriptomics [22], and for any Hydra gene of interest the spatial and cell-type expression pat-
terns are now available on the HydrAtlas server (https://hydratlas.unige.ch/blast/blast_link.cgi) [23]. Specific
signatures were obtained by analyzing the expression patterns of (1) neuropeptides such as RGamide,
RFamide, KVamide, or LWamide [24–26], the neuropeptide Hym-355 enhancing neuronal differentiation [3], (2)
86 genes identified in a microarray screened with cRNAs from nerve-less animals [27], (3) neurogenic genes
that regulate the proliferation and/or the differentiation of progenitors such as CnASH [17, 28], prdl-a [23],
COUP-TF, prdl-b [29], hyZic [30], cnox-2 [22], CREB [29], Myc1 [31, 32], FoxN1, PaxA, PaxB, Pou4F2 [22], (4)
the two Piwi proteins (HyWi, HyLi) strongly expressed in ISCs and nematoblasts [33], (5) some gap junction
proteins, innexin-2 being involved in neurotransmission [34]. No pan-neuronal marker was identified in Hydra
yet. As early markers of nematocyst differentiation, the minicollagens N-COL1 and NOWA [35, 36] are compo-
nents of the wall, spinalin of the spines [37], N-COL15 of the tubule [38], nematogalectins A and B of the tubule
from distinct types of nematocysts [39]. Finally, a proteomic analysis of the venom identified 410 secreted
proteins in nematocysts [40] (not shown here)
Fig. 3 The homeoprotein prdl-a as a marker of apical neurogenesis. (a) Double immunostaining of BrdU (green) and
prdl-a (red) expressed in apical neuronal progenitors or nerve cells of homeostatic animals (upper panel) or animals
having regenerated their head (lower panel). All animals were incubated with BrdU for 4 h and then washed out to
remain intact or to undergo mid-gastric bisection (red arrow). White arrow heads indicate the prdl-a+/BrdU+ cells,
which are more numerous in the regenerated than in the homeostatic head. (b) De novo neurogenesis evidenced
by prdl-a (red) immunostaining in the presence (top panel) or the absence (lowerpanel) of interstitial progenitors
after hydroxyurea (HU) treatment. The animals were exposed or not to 10 mM HU in three rounds (2× 24 h and
1× 32 h). After the third HU treatment, animals were transferred to HM, bisected (red arrow) and let to regenerate
for seven days. Note the absence of prdl-a+ cells in HU-treated animals. Nuclei were counterstained with DAPI
(blue). Image acquisition was done on a Leica LSM700 confocal microscope. Scale bar: 100 μm
8 Wanda Buzgariu et al.

Fig. 4 Loss of terminal neuronal differentiation in Hydra undergoing apical or basal regeneration in the absence
of ISCs or interstitial progenitors. HU treatment and regeneration were performed as in Fig. 3. The apical (a)
and basal (b) nerve nets were identified after RFamide immunostaining (green) and nuclear DAPI counterstain-
ing (purple). Note the sparse RFamide+ nerve cells in apical (a) and basal (b) regions regenerated after HU
treatment. Image acquisition was done on a Leica LSM700 confocal microscope. Scale bars: 100 μm

force-fed, pointing to an important morphogenetic role played by

the epithelial cells [46].
The Hydra Peptide Project identified about 500 peptides,
half neuropeptides, half epitheliopeptides, with specific roles in
neurotransmission, morphogenesis, and differentiation [48, 49].
However, not much is known concerning the role and the regu-
lation of the neuronal-epithelial cross talk on the slow and fast
modes of neurogenesis in intact and regenerating animals, respec-
tively. These questions require the monitoring of the expression of
neuronal markers in proliferating and differentiating progenitors as
well as in fully mature nerve cells (Fig. 5).

1.2 Principles The Hydra nervous system is well visualized with the immunos-
of Immunodetection taining procedures applied either on whole animals or on cells that
on Whole-Mount keep their morphology after tissue maceration [41]. In both con-
and Macerated texts, the procedure is based on the specific antigen–antibody rec-
Tissues in Hydra ognition followed by different detection techniques to visualize
the cells that express or overexpress a known antigen. The immu-
nodetection procedure follows the classical steps: fixation and per-
meabilization of the tissue, saturation of unspecific sites, and
antigen recognition by a specific antibody followed by detection
and visualization. The expression patterns obtained in intact or
regenerating Hydra as well as in specific cell types can also be veri-
fied at the transcriptomic level on the HydrAtlas server (https://
hydratlas.unige.ch) [23].
 Methods to Monitor Adult Neurogenesis in Hydra 9

Fig. 5 BrdU immunostaining combined with Hym-355 detection in homeostatic animals. The neuropeptide
Hym-355 is expressed in a subset of apical and basal neurons. The animals were exposed to BrdU for 4 h,
washed and subsequently maintained in HM for 2 days and then fixed. After the in situ hybridization procedure
to detect Hym-355 expressing nerve cells (a, purple), the animals were immunolabeled for BrdU (b, c, green).
In homeostatic condition, only few apical neurons do express Hym-355 and are positive for BrdU (arrows). The
bright field (a) and fluorescence (b) images were acquired with a Leica DM5500 fluorescence microscope. Box
in (b) corresponds to panel (c). Scale bars: 100 μm (a, b) and 20 μm (c)

1.2.1 Immunodetection The whole-mount immunodetection procedure is a robust method

on Whole-Mounts to detect with specific antibodies the spatial or temporal expression
pattern of neurogenic proteins or neuropeptides. In complement
the same procedure can be used to detect with antibodies raised
against tagged material incorporated into macromolecules such as
Bromodeoxyuridine (BrdU) incorporated into newly synthesized
DNA [50] or digoxygenin (DIG)-labeled riboprobes that bind
transcripts [51] (Fig. 5). As a consequence, immunodetection
allows the visualization of proteins, DNA, and transcripts in tissues
that show a preserved architecture.

1.2.2 Immunodetection To get a more accurate analysis at the cellular level, the immuno-
on Cells Obtained detection procedure can be performed on histological sections
After Tissue Maceration [18, 52] or on cells directly fixed on slides after tissue maceration
[41]: In the presence of acetic acid and glycerol, the tissues are dis-
sociated into single cells or small clusters, which are subsequently
fixed with paraformaldehyde (pFA) that preserves their typical cel-
lular architecture. Hydra-specific or exogenous epitopes as BrdU
can next be immunodetected. The maceration technique can be
adapted for small amount of tissue such as head- or foot-­
regenerating tips [4]. The strengths of this method are multiple: it
identifies cell types and specific subcellular localization, it allows
the precise quantification of each expressing cell type, including
neurons, interstitial cells or clusters of nematoblasts (Fig. 1b).

1.2.3 Antibodies As mentioned above, the Hydra nervous system produces

numerous neuropeptides. Since 1982, their immunodetection
on intact animals proved to be extremely useful to identify the
anatomical organization and localization of specific subsets of
neurons in Hydra [53, 54]. The vasopressin-like or RFamide
sera were critical to monitor the phenotypic conversion of neu-
rons as they get displaced along the body axis [10]. With the
10 Wanda Buzgariu et al.

development of bioinformatic tools and genomics, evolution-

arily conserved regions can be easily mapped and commercially
available antibodies raised against such mammalian epitopes can
be used to cross-react against Hydra proteins. Alternatively
monoclonal [55] or polyclonal antibodies can be raised against
Hydra proteins, either lab-made such as the sera against the
RFamide neuropeptide [53], the transcription factor CREB
[56], and the homeoprotein prdl-a [18] or commercially pro-
duced. The most efficient way to validate the specificity of a
given antibody is to perform gene silencing, usually through
RNA interference in Hydra, to assess the decrease in protein
abundancy by Western analysis or immunofluorescence [57].

1.2.4 Tissue Fixation Fixation and permeabilization are crucial steps as they allow the
and Tissue preservation of cells and tissue architecture. Paraformaldehyde (pFA)
Permeabilization and formaldehyde (FA) are two cross-linking agents commonly used
as fixatives; they ensure a good penetrability and preserve the anti-
gens by forming chemical bonds between the proteins. In contrast,
alcohols such as ethanol and methanol, or acetic acid are precipitat-
ing fixatives that disrupt the hydrophobic bonds and thus denature
the tertiary structure of proteins. In fact, ethanol or methanol allow
a good permeabilization by precipitating membrane proteins, lead-
ing to the formation of pores in the membrane, thus contributing to
the leakage of RNA or cytoplasmic proteins and to a poor cellular
preservation. Therefore, the commonly used method to fix Hydra
polyps for whole-mount immunodetection or in situ hybridization
(ISH) combines pFA and alcohol fixatives. As an alternative the
Lavdowsky fixative that combines ethanol, FA, acetic acid and water
(50/3.7/4/42), also takes advantage of the properties of cross-link-
ing and precipitating agents [55]. A variant of the Lavdowsky fixa-
tive that does not contain acetic acid is often used to detect nuclear
antigens. Finally, the mercury-containing “Helly” fixative or the
Zinc–FA fixative used on insect brains to preserve morphology and
improves immunodetection at synapses [58], can be used efficiently
when the other fixatives fail. For each new epitope/antibody several
fixatives need to be tested together with the fixing conditions such
as temperature (4 °C, 18 °C, 37 °C) or duration of fixation. Tissue
permeabilization is usually completed by adding a detergent such as
Triton X-100 or Tween 20, again applied for a period of time that
needs to be adapted to each antigen of interest.

1.2.5 Antigen Detection The detection of an antigen can be made directly with the primary
antibody or indirectly through a secondary antibody that binds to
the primary one. The indirect method is preferred as Hydra spe-
cific antibodies conjugated with fluorochrome are rare and immu-
nodetection is more sensitive when indirect as the signal is
amplified. A large choice of secondary antibodies coupled to
fluorochromes or enzymes are available. Amplification is optimal
 Methods to Monitor Adult Neurogenesis in Hydra 11

when a Tyramide detection system is used with conditions adapted

for each antibody (see supplemental figure 1 in ref. 21). Unspecific
binding of antibodies to reactive sites of proteins is blocked by BSA
(bovine serum albumin) or normal serum from the species used to
raise the secondary antibody. A common counterstaining is made
by nuclear staining, which allows a better cell-type and tissue-layer
identification through the shape and size of nuclei.

2 Materials

All stock solutions are prepared with MilliQ water in sterilized bot-
tles with screw-cup, then autoclaved or filtered through a 0.22 μm
Steritop filter, and finally stored at 4 °C or at room temperature
(RT) depending on the buffers.
After opening, the stock solution should be checked regularly
to be discarded before they become viscous or turbid, with visible
signs of contamination. Experiments are always performed with
fresh solutions, i.e. 10× stock solutions freshly diluted in sterile
dishware with MilliQ water to prepare the requested amount of 1×
solution for a single experiment.

2.1 Hydra 1. Hydra medium (HM): 1 mM NaCl, 1 mM CaCl2, 0.1 mM

Maceration KCl, 0.1 mM MgSO4, 1 mM Tris pH 7.6 [59]. HM is pre-
pared from three stock solutions that can be stored several
weeks at RT once autoclaved:
For the stock solution A (0.5 M Tris, 500×) dissolve 60.57 g
Tris-base in 900 ml H2O, adjust the pH to 7.7 and complete
the volume to 1000 ml. For the stock solution B (1 M MgSO4,
10,000×) dissolve 61.6 g MgSO4⋅7H2O in 250 ml H2O. For
the stock solution C (0.5 M CaCl2, 0.5 M NaCl, 0.05 M KCl,
500×) dissolve 54.77 g CaCl2⋅6H2O, 14.6 g NaCl, 1.85 g KCl
in 500 ml H2O. Autoclave the solutions A, B, and C. To pre-
pare 1 l HM solution, dilute 2 ml solution A, 100 μl solution
B and 2 ml solution C in 1000 ml H2O.
2. Macerating solution (MS): 7% acetic acid, 7% glycerol in
H2O. Add 0.7 ml glycerol and 0.7 ml glacial acetic acid to
8.6 ml H2O. Use the fume hood for preparation. Do not auto-
clave. Store at RT no longer than 2 weeks.
3. 10% Tween 80: Add 1 ml Tween 80 to 9 ml H2O (see Note 1).
Do not autoclave. Keep the solution for 1 month at RT.

2.2 Fixation 1. 4% Urethane: Dissolve 1 g urethane in 25 ml HM. Store at

4 °C. Always wear gloves as urethane is carcinogenic.
2. Lavdowsky fixative: 50% ethanol, 3.7% FA, 4% acetic acid. Add
1 ml 37% FA to 5 ml ethanol, 0.4 ml acetic acid and 4 ml
H2O. Prepare it fresh, do not store.
12 Wanda Buzgariu et al.

(a) Lavdowsky fixative without acetic acid: 50% ethanol, 3.7%

FA. Add 1 ml 37% FA to 5 ml ethanol and 4 ml
H2O. Prepare it fresh and do not store.
(b) 8% Paraformaldehyde (PFA): Dissolve 8 g pFA in 80 ml
HM preheated at 65–70 °C in a glass bottle and stir the
mixture on a water bath maintained at 70 °C under the
hood. Add 100 μl 1 N NAOH and stir the solution until
the solution becomes clear. After cooling to RT, adjust the
volume to 100 ml with HM and adjust the pH to 7.5 with
0.1 N NaOH. Filter the solution using a steriflip filter.
Store at 4 °C for 2 weeks maximum (see Note 2).
(c) 4% PFA: Dilute 50 ml 8% pFA with 50 ml H2O and read-
just the pH to 7.5 with 0.1 N NaOH.
(d) 3.7% Formaldehyde (FA): Add 1 ml 37% FA to 9 ml H2O.

2.3 BrdU BrdU (5-Bromo-2'deoxyuridine is an analog of thymidine that is

Immunodetection rapidly incorporated into DNA during the replication phase of the
cell cycle.
1. BrdU solution (10 mM): Add 154 mg BrdU to 50 ml HM in a
50 ml centrifugation tube, protect the tube from light and stir
the solution that can be stored at 4 °C for a few days (see Note
3).
2. 10× PBS buffer: 1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4,
11 mM KH2PO4. Dissolve 80 g NaCl, 2 g KCl, 29 g Na2HPO4
12H2O and 2 g KH2PO4 in 1000 ml H2O. Verify that pH is
6.8. Autoclave and keep at RT (see Note 4).
3. PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.1 mM
KH2PO4. Dilute 50 ml 10× PBS to 500 ml (see Note 4).
4. PBST: 0.5% (v/v) Triton X-100 in PBS. Add 0.5 ml Triton
X-100 to 100 ml PBS (see Note 1).
5. Blocking solution: 2% BSA. Dissolve 1 g BSA in 50 ml PBS and
stir until the solution becomes clear. Filter through a Steriflip
and store at 4 °C (see Note 5).
6. 2 N HCl: Add 2 ml 37% HCl to 10 ml of H2O (see Note 6).
7. DAPI (4,6-diamidino-2-phenylindole) stock solution (1 mg/
ml): Dissolve 10 mg DAPI in 10 ml H2O. Aliquot and store at
−20 °C.
8. DAPI working solution (1 μg/ml): Dilute 1 μl DAPI 1 mg/ml
into 1 ml PBS.

2.4 Antibodies 1. The polyclonal anti-prdla antibody was produced in rabbit

after immunization with the N-terminal fragment of prdl-a
fused with 6HIS [18]. The polyclonal anti-RFamide antibody
was produced in rabbits and kindly provided by Cok
 Methods to Monitor Adult Neurogenesis in Hydra 13

Grimmelikhuijzen [53]. For BrdU immunodetection the

BrdU labeling and detection kit from Roche (ref 11444611001)
provides the most reliable results. Dilute the antibody 1:20 in
the labeling solution found in the kit (see Note 7).
2. The optimal titer for each primary antibody should be estab-
lished after testing a series of dilutions; too high concentra-
tions increase the background staining as the antibody
accumulate at the cell or tissue surface, too low concentrations
provide low-­intensity signals due to a reduced antigen–anti-
body interaction.
3. In the indirect immunostaining procedure, the secondary anti-
body needs to be chosen according to the available filters and
lasers that equip the fluorescent/confocal microscopes. If a
double antigen detection is required, select the secondary anti-
bodies considering the possible cross talk between the excita-
tion and emission wavelengths of each fluorochrome.

2.5 Mounting The samples should be mounted in a medium adapted for fluores-
Medium cence, i.e. characterized by a high refractory index, no autofluores-
cence and protecting against photobleaching. The MOWIOL
mounting medium (polyvinyl alcohol) fulfills these criteria. It is a
widely used lab-made preparation, less expensive than the com-
mercially available ones.
1. 0.2 M Tris pH 8.5: Dissolve 2.42 g Tris base in 100 ml of
H2O. Adjust in a fume hood the pH to 8.5 with approximately
710 μl of 37% HCl solution.
2. Add 24 g MOWIOL 4–88 to 60 g glycerol and stir well.
Subsequently add 60 g H2O and mix well for another 2 h at
RT.
3. Add 100 ml 0.2 M Tris pH 8.5 and continuously stir at 52 °C
on a heating plate until the powder is completely dissolved.
This might take 4–5 h.
4. Dispatch the mix in 50 ml centrifugation tubes and centrifuge
at 5000 × g for 15 min.
5. Carefully collect the supernatant and aliquot into 15 ml coni-
cal tubes.
6. Store the Mowiol mounting medium at −20 °C up to 1 year.
For current use, keep aliquots at 4 °C.

2.6 Equipment 1. Stereomicroscope.

2. Pasteur glass pipette.
3. 360° vertical rotator.
4. Shaker.
5. Superfrost Plus microscope slides (Thermo Scientific, Gerhard
Menzel).
14 Wanda Buzgariu et al.

6. Coverslips: 22 × 40 mm, 0.13–0.17 thickness.

7. Surgical scalpel N°3.
8. Surgical blades N°10 (Ruttgers Solingen).
9. Plastic Petri dishes (6 and 9 cm diameter).
10. Silicon bulbs, 5 mm diameter for Pasteur pipette.
11. Steriflip and steritope for filtration (Millipore).
12. Hydrophobic pen (DAKO Pen).
13. Slide staining tray, Simport Scientific.

3 Methods

3.1 Hydra Tissue 1. Collect five intact animals in a 1.5 ml Eppendorf tube with the
Maceration Protocol help of a Pasteur pipette. Remove the medium and replace  it
and Fixation with 400 μl of fresh HM, repeat the washing 2–3× times (see
Note 8).
2. After the last wash, eliminate carefully all the liquid and add
100 μl MS. Let the tube on a rack and mix gently, from time
to time, until the tissue dissociates into a homogenous cell sus-
pension (see Note 9).
3. After 30–60 min, fix the cell suspension by adding 100 μl 8%
pFA, mix gently and let incubate for 1 h at RT.
4. Add 10 μl of 10% Tween 80 and mix gently.
5. With the DAKO PEN, label a square of about 25 × 20 mm on
a Superfrost Plus slide. Let it dry for about 10 min.
6. Add the cell suspension drop by drop on the surface delimi-
tated by the hydrophobic marker lines.
7. Let the slides dry for about 2 days at RT.
8. The slides are ready to be processed or can be stored into a box
at −20 °C.

3.2 Hydra Fixation 1. Collect 15–20 intact animals with a glass Pasteur pipette in a
for ISH and Whole-­ graded 2 ml Eppendorf tube and adjust the final volume to
Mount 1 ml HM.
Immunostaining 2. Add 1 ml 10 mM BrdU solution and transfer the animals in a
plastic Petri dish (6 cm diameter) that contains 10 ml 5 mM
BrdU solution. Protect from light and incubate for the desired
period of time (see Note 3).
3. To wash out BrdU, collect the animals in 2 ml tube and wash
them several times in HM by gently aspirating the liquid and
replacing it with fresh medium.
 Methods to Monitor Adult Neurogenesis in Hydra 15

4. To initiate regeneration, transfer the animals in a plastic Petri

dish (9 cm diameter) in 50 ml HM and place the dish under
the stereomicroscope. Let the animals relax for a few minutes.
5. Bisect the animals with a scalpel at half of the body column
length and transfer the head regenerating halves to a new Petri
dish filled with 50 ml HM. Let the animals to regenerate (see
Note 10).
6. Collect 15–20 intact or regenerated animals in a graded 2 ml
Eppendorf tube and wash them several times with HM as
described at step 3.
7. Adjust the final volume to 1 ml. Let the animals to relax a few
minutes.
8. Immediately before fixation, add 1 ml 4% urethane to reach a
2% final concentration. Mix gently and wait for 60 s until the
animals are completely relaxed (see Note 11).
9. Remove 1 ml urethane solution and immediately add 1 ml 8%
pFA to reach a 4% final concentration. Mix well and let the
animals fall to the bottom of the tube and aspirate the maxi-
mum amount of the liquid.
10. Wash 3–4 times with 4% PFA by eliminating each time the
liquid and replacing it with fresh fixative.
11. Place the tubes on a shaker/rotator and fix the animals for 4 h
at RT (see Note 12).
12. Aspirate the PFA and replace it with ethanol 100%; replace
several times the ethanol until the brown pigment is
solubilized.
13. Store the fixed samples at −20 °C in ethanol until the ISH
procedure.

3.3 Hydra Fixation 1. For BrdU incubation and animal fixation, proceed as in
for BrdU Subheading 3.2 from steps 1 to 8.
Immunostaining 2. Remove the maximum amount of the urethane solution and
on Whole-Mount replace it with Lavdowsky fixative without acetic acid. Wash
several times with the fixative.
3. Place the tubes on a shaker or rotator and let the animals fix
either for 1 h at 37 °C or overnight at 4 °C (see Note 12).
Proceed immediately after fixation with the immunodetection.

3.4 Immuno­ The immunodetection procedure is performed in a dark humidity

detection of Hydra chamber (see for example the StainTray slide staining system pro-
Cells After Tissue posed by Sigma). During antibody incubation, the atmosphere is
Maceration kept humid by adding PBS in the bottom of the chamber.
16 Wanda Buzgariu et al.

1. Rehydrate the cells by washing 3× 10 min with PBS (see

Note 13).
2. Incubate the cells with 0.1% PBST for 30 min (see Note 14).
3. Add the blocking solution for 1 h at RT (see Note 15).
4. Dilute the primary antibody as desired in blocking solution.
5. Gently remove the blocking solution and add 100 μl of ­primary
antibody solution. Incubate overnight at 4 °C or alternatively
3–4 h at RT (see Note 16).
6. Wash the slides 4× 10 min with PBS.
7. Dilute the secondary antibody in PBS as recommended by the
supplier.
8. Remove PBS, add the secondary antibody solution and incu-
bate for 2–4 h protected from light.
9. Wash the slides 4× 10 min with PBS.
10. Incubate with 1 μg/ml DAPI for 10 min (see Note 17).
11. Wash the slides 2× 5 min with PBS.
12. Wash the slides fast with H2O and let them dry at RT
for 15–20 min protected from light.
13. Once dried, add one drop of mounting medium and apply the
coverslip by holding it at 45 °C. Gently descend the cover-
slip allowing the mounting medium to cover the surface
delimitated by the square. Avoid producing air bubbles (see
Note 18).

3.5 Immunostaining The procedure presented below is a general protocol, which has to
on Whole-Mount be adapted for each antibody. Unless specified, the incubation
Hydra steps are performed at RT in 1.5 ml Eppendorf tubes. The proto-
col should be adjusted according to the fixative. If samples are
freshly fixed with Lavdowsky fixative, with or without acetic acid,
start with step 1. If samples are stored in ethanol after pFA fixa-
tion, directly proceed to step 2.
1. Remove the Lavdowsky fixative and replace it with PBS. Repeat
quickly the washing step twice. Proceed to step 3.
2. Rehydrate the pFA-fixed animals stored in ethanol by washing
successively in 75%, 50% and 25% ethanol, each step for
5–10 min (see Note 8).
3. Wash 4× 10 min with PBS.
4. Remove PBS, add PBST to permeabilize for 30 min (see Note
14).
5. Optional for nuclear antigen detection. Otherwise proceed to
step 7. Remove PBST and incubate in 2 N HCl for 30 min (see
Note 19).
 Methods to Monitor Adult Neurogenesis in Hydra 17

6. Remove HCl and wash quickly 6× in PBS to eliminate all HCl

traces. Complete with 2× 5 min washes in PBS (see Note 20).
7. Add the blocking solution for 60 min at RT (see Note 15).
8. Dilute the primary antibody as desired in blocking solution.
9. Remove the blocking solution, add at least 100 μl primary
antibody solution. Incubate overnight at 4 °C or alternatively
3–4 h at RT (see Note 21).
10. Remove the primary antibody, wash quickly twice in PBS, then
4× 10 min.
11. Dilute the secondary antibody in PBS as recommended by the
supplier.
12. Remove PBS, add the secondary antibody solution and incu-
bate for 2–4 h protected from light (see Note 22).
13. Remove the secondary antibody, wash quickly twice in PBS,
then 4× 10 min.
14. Remove PBS and add 300 μl 1 μg/ml DAPI for 10 min (see
Note 23).
15. Wash quickly twice with PBS, then 2× 5 min.
16. Wash quickly with H2O.
17. Mount the animals on glass slide with the mounting medium.
Gently descend the coverslip allowing the mounting medium
to cover the surface. Avoid producing air bubbles (see
Note 24).

3.6 Immunostaining The immunodetection protocol described above can be applied to

on Whole-Mount samples previously processed for in situ hybridization (ISH) to
Hydra After In Situ combine the detection of both protein and gene expression. Since
Hybridization the 1990s, gene expression patterns can be investigated in whole-­
mount Hydra. The classical procedure comprises fixation of the
samples, hybridization of Digoxygenin (DIG) or Fluorescein-­
labeled riboprobes to the targeted transcript, and immunodetec-
tion of these hybridized riboprobes with an anti-DIG or an
anti-Fluorescein antibody coupled to alkaline phosphatase, fol-
lowed by a colorimetric detection of the coupled enzyme with
NBT/BCIP substrate (for a detailed protocol, see ref. 53). The
presentation of the complete ISH procedure is omitted here and
only the steps that link the two methods are detailed.
1. At the end of the ISH procedure, stain the samples with NBT/
BCIP as in [51] and follow carefully the development of the
staining. Once the pattern is obtained, wash out as indicated
the NBT/BCIP substrate to block the staining.
2. Post-fix the samples in 3.7% FA for 30 min at RT.
3. Wash out FA by rinsing two to three times with methanol.
Another Random Scribd Document
with Unrelated Content
The Project Gutenberg eBook of Sales
Resistance
This ebook is for the use of anyone anywhere in the United States
and most other parts of the world at no cost and with almost no
restrictions whatsoever. You may copy it, give it away or re-use it
under the terms of the Project Gutenberg License included with this
ebook or online at www.gutenberg.org. If you are not located in the
United States, you will have to check the laws of the country where
you are located before using this eBook.

Title: Sales Resistance

Author: Henry Still

Illustrator: Ed Emshwiller

Release date: May 14, 2019 [eBook #59505]

Language: English

Credits: Produced by Greg Weeks, Mary Meehan and the Online

Distributed Proofreading Team at http://www.pgdp.net

*** START OF THE PROJECT GUTENBERG EBOOK SALES

RESISTANCE ***
sales resistance
 BY HENRY STILL

When Consumption means prosperity, when

the
Pulitzer Prize is awarded to advertising copy,
when the Salesman is the most respected
citizen
in the land.... What chance has a non-
consumer?

[Transcriber's Note: This etext was produced from

Worlds of If Science Fiction, August 1956.
Extensive research did not uncover any evidence that
the U.S. copyright on this publication was renewed.]
On his way home from the concert, Perry Mansfield whistled a
pleasant melody from an old Stravinsky classic. But then, troubled by
his conscience and that of his psychiatrist, he stopped to study the
program again.
What was that modern symphony? Oh yes, "The Flivver". The music
was supposed to have its roots in antiquity when someone started
converting the metal wealth of the earth on an assembly line. Those
screeching noises were drill presses and lathes and automatic
hammers. The syrupy melody was the saintly salesman who
disbursed the wealth of gadget and machine like melted butter across
the bread of the land.
Perry tried to like it. But he didn't. And that disturbed him. It meant
his psychotherapy wasn't working. Dr. Stone would run him through
the mechanical analyzer again and scold over the results.
His simple act of walking home instead of riding an anti-gravity
putterseat labeled him as a misfit. But it seemed silly to rent a flying
stool just to travel two blocks.
The fact was, he liked to walk.
Perry sighed, discouraged, as he waited for the fluorescent scanner
to identify his insides and open his front door.
It opened. The lights came on. Recorded music, somewhat tuned to
his mood, poured from concealed amplifiers.
And then he noticed the note clipped to the door.
His hand trembled as he took it down. The beautiful pastel gray of
the enclosing envelope was an anachronism itself, and therefore
marked unmistakably its almost priestly origin.
The platinum engraved card inside said simply:
A Master Salesman has chosen you for his next call.
Perry placed the note carefully on a plastic table. He inhaled deeply
and held it for a moment to steady his nerves.
A Master salesman. No one of that stature had ever called upon him
before. It was an honor like—like a mayor or a bishop. It meant he
had attained top level on the universal measuring stick—an A-
number-1-plus-plus credit rating.
The prospect should have saturated him with pleasure. But, like the
sharp new music, it didn't.
This card also meant he was expected to buy something. Something
big and expensive. And he didn't want or need something big and
expensive.
He wished they'd leave him alone.
Perry clapped his hand to his mouth as though someone might have
heard the thought.
What was wrong with him anyway? He wasn't a recluse. He wanted
to indulge and enjoy the polished luxury of his world. He wanted to
be conventional. He was young and handsome and tall and dark. He
had a good job. He had a pleasant and comfortable legal
arrangement with a girl in the next block.
But truly, what he had was all he wanted.
He glanced at the card on the table. He could always say no. It
wouldn't be easy, but he could say no.
Perry thumbed through the Pulitzer prize winning work for 2087
which had been delivered yesterday as part of his book club
subscription. He had seen it already, of course, in a dozen magazines
and a hundred copies of his facsimile newspaper. It was the
advertising copy for Cor-T-Zan foundation garments. But he didn't
need a corset and the spartan simplicity of the fragile, lovely words
bored him.
He switched on television. A phrenetic band was hammering out the
new top jingle on the Hit Parade:
 Tootsie gum, tootsie gum
Ooh yum-yum, it's touched with rum;
Love that girl with eyes so hot,
TOOTSIE GUM hits the spot.

Perry switched off the set.

He was alone. He could be honest with himself. The whole damned
business irritated him. If he was out of step with the times, to hell
with the times.
Mr. Master Salesman didn't even say when he would call. You were
expected to sit on the edge of your chair, waiting for the great man
to appear.
Finally Perry decided what he'd do. He'd simply not open the door
when the MS came knocking.
Upon that decision, he slept well.

Sometime later he dreamed of frying bacon over an open fire in the

woods, although he hadn't been out to the park in three years.
When he opened his eyes, the sun was up. He still smelled bacon
frying.
Perry crawled out of bed, fumbled into his robe and followed his nose
to the kitchen-bar.
There, in his favorite chair, sat a handsomely-dressed, distinguished
man with florid complexion, iron gray hair and a fashionable paunch.
Strips of bacon were frying on the bright, spotless steel of the
cooking shield.
"How did you get in here?" Perry asked crossly.
"Serve-All does all," his visitor said cryptically and smiled the smile
that's known around the world. Perry would have no opportunity to
shut out the Master Salesman. He was in.
"You are Mr. Mansfield?"
"Yes sir," Perry said, uncertain of decorum.
"My name is Marlboro," the MS said in melodius tones. "Master is the
proper term used in addressing us. Please sit down."
"Yes, Master," Perry said. He felt like a fool and sat down.
"Breakfast will be served in a few moments," Marlboro said. "I hope
you don't mind, I examined your excellent library before you came
in." He pulled a volume off the shelf. "This is a beautiful old first
edition. Wherever did you find it?"
It was Perry's copy of "Basic Sales Techniques" with Burton footnotes
on vacuum cleaner sales charts for the last half of the 20th century.
"I've read it, of course," Marlboro continued, "but I've never owned a
copy." He caressed the dogeared cardboard cover. "Isn't it fantastic?
In that barbaric century the customers sometimes refused to buy
from our predecessors in the Guild. It seems impossible that anyone
could have been so crude as to turn away one of those sturdy
pioneers at the door."
Perry shifted uncomfortably. He had been prepared to turn away one
of the Great Men at his door.
"Ah!" Marlboro exclaimed. "Your bacon is ready, young man."
At a flick of the salesman's finger, the golden strips of meat lifted into
the air and floated to an absorbent mat on the table. Perry stared.
Not a bubble of fat had fallen to the floor in passage.
"How did you do that?"
"Serve-All does all," the MS said coyly.
"Mine doesn't," Perry said.
"Of course not!" Marlboro moved deftly into the opening. "You need a
new one."
So he had tumbled for the first trap. Perry blushed and ate a piece of
bacon.
The Master hefted an object to the table top. It was a hemisphere
about 18 inches in diameter, smooth and featureless except for a
handle on the curved top. It was painted psychological green.
"This is the new Serve-All," Marlboro said glibly. "Notice its smooth
unobtrusive shape. No working parts exposed, but inside is a mass of
circuits and servos around a baby reactor ready to do everything for
you."
"The bacon," Perry persisted. "How?"
He was aware that the first step in successful selling is to arouse
curiosity. But he was confident he could refuse to buy, though it be
contrary to convention and good taste.
"Fingers of energy," the MS said. "Invisible, sensitive fingers of
energy reach out of here—" He tapped the Serve-All dome. "—and
they'll do anything that needs doing, at your mental command. Right
now this one's tuned to me, but a minor adjustment will fit it to your
personal needs. Here, let me show you something else."
Perry felt a gentle, firm pressure on his left cheekbone. It moved
down his cheeks, across his upper lip and up the other side. Then
under his chin.
Marlboro whipped out a pocket mirror.
Perry had just been shaved.
"See?" the Master beamed. "Wonderful isn't it?" Perry nodded. That
was calculated to put him in a yes mood.
While they talked the Serve-All cleared the breakfast clutter and
cleaned the cooking shield without visible remains or waste. Marlboro
pulled a contract pad out of his pocket.
"I presume I can put you down for one of these."
"I don't need it," Perry said. "My old one is good enough."
"Ridiculous!" Marlboro said indignantly and then chuckled good-
humoredly. "Oh, I see what you're doing. You're trying some of the
old tricks from the 20th century. Well, I like a game of wits, too. Look
what else this model will do."
While Perry watched, the Serve-All repaired a broken knob on a
plastic chest, cleaned the rug and etched a mural of a voluptuous
nude on one blank wall.
"If you'll excuse me," Perry murmured, "it's time for me to go to
work."
"Of course, of course," the Master laughed jovially.
In rapid succession a comb dressed Perry's hair, his robe and pajamas
were whisked off and his street clothes came floating out of the
closet on more invisible fingers of energy.
Before he knew it, he was ready for work.
"I really must be going, too," Marlboro said, "if you'll just sign here."
"How much is it?"
The Master Salesman sighed.
"You're really very difficult. It's $9,785, plus tax."
"I can't afford it."
"Now, Mr. Mansfield. A joke's a joke. If your credit rating wasn't the
finest, I wouldn't be here. I know, and you know, your income is
mortgaged for only 15 more years and your life expectancy is at least
50."
Perry moved uneasily toward the bathroom. An invisible finger of
energy opened the door for him.
"If you don't mind," he said angrily, "this is something I'm quite
capable of doing for myself." He slammed the door.
But the Serve-All flushed the toilet for him.
When he emerged, Marlboro's patience also was gone.
"Sign," he said firmly.
"I don't want it."
"Young man," the Master said thinly. "You don't realize what
dangerous ground you're on. If you do not cease this rudeness at
once, I'll report you to the council."
"Report and be damned! I don't need your gadget and I'm not going
to buy it. Now get out!"
Marlboro was blue with rage. He backed uncertainly toward the door
and stopped.
"This borders on sacrilege," he whispered. "You'll hear from me
again. Soon."
Perry slammed that door, too, and walked jauntily to work.

He heard from the Master Salesman again—exactly two hours later.

The message tube delivered a summons ordering him into City Court.
That afternoon.
Perry went. He had never been in court before. He was frightened
and regretful that he had been so abrupt with Marlboro. But he
resented the invasion of his privacy and to bolster his courage, he
built that anger into a fair rage by the time he reached the
courtroom.
Marlboro was there. A judge was there. And on each of two tables
squatted a metal box with voice tubes. A bailiff guided him to his
table and placed the voice tube in his hand.
"You're late Mr. Mansfield," the judge snapped. "Justice must be swift
and you're impeding it." He lifted a printed card and scanned it near-
sightedly for a moment. "You're here charged with violating the
public interest by failing to purchase an item which you are able to
consume and which you can afford to buy."
"There's no law against—" Perry began indignantly.
"Don't tell me your troubles, young man," the judge interrupted.
"That's what your lawyer's for." His gesture indicated the metal box.
Perry held the voice tube dumbly. The bailiff leaned over his shoulder.
"You tell your side of the story in there," he whispered.
Marlboro was muttering rapidly and at great length into his "lawyer."
Perry did likewise, relating all he could remember of the morning
fiasco. When he finished, the machine whirred, whistled and
harrumphed twice before spewing out several yards of perforated
tape.
The plaintiff's counsel did the same, except the tape was longer.
"Now Mr. Bailiff," the judge said, "you may bring in the jury."
Perry was no longer surprised when the jury was rolled in. It was a
large gray analog computor mounted on wheels. The judge stepped
down from the bench and fed in the two conflicting tapes.
The jury digested the information noisily.
"It's an old model," the judge apologized, but just then a white card
popped out on a small metal tray. The bailiff delivered it to the judge.
He studied the card. Perry's heart thumped painfully during the
calculated period of suspense.
"As you attempted to inform the court earlier, Mr. Mansfield," the
judge said somberly, "there is no law in the land which forces you to
buy any item from our distinguished colleagues of distribution."
Perry's heart brightened and he slid back from the edge of the chair.
"However," the judge peered down, "it has been held by many courts
that when the public interest is to be served by the individual
purchase of a piece of merchandise which that individual can
consume and which that individual is able to buy without financial
hardship, then that individual must sacrifice his emotional reluctance
to the good of society."
The jurist paused thoughtfully.
"I think, Mr. Mansfield, that you should relearn the basic tenets of our
society and economy. First, Consumption is Prosperity and that
derives from the ancient law of Supply and Demand. S & D means, in
simple terms, that when there is a supply of something, a demand
must be created to consume it. That is why we have Master
Salesmen. That is why they are the staunchest and most highly-
respected citizens in our land."
He bowed to Marlboro who assumed a benevolent smile.
"This court decrees," the judge said sternly, "that you are to purchase
an item known as the 2087 Serve-All from Master Salesman Marlboro
and customary steps will be taken to attach your future salary to
satisfy the stipulated payment schedule. Court dismissed."
Perry was too stunned to move. His petty rebellion had collapsed into
a pot of embarrassment. He was vaguely aware of Marlboro shaking
his hand with a moist, jovial palm.
"No hard feelings, young man," the MS said. "It was really quite
interesting. I haven't had a case like this in five years."
The condescension stirred Perry's anger again.
"I demand an appeal!"
The judge was leaving the bench, but he turned back.
"Appeal bond is $2000."
"No appeal," Perry said glumly.

He walked home. The 2087 Serve-All was there waiting for him, in
the middle of his living room floor.
Marlboro had tied a gay red ribbon around it to cheer him.
He wasn't cheered. The thing must have been delivered even while
he was in court. There had never been a doubt that he would lose
the case. Rage began to crawl its acid path through his stomach
again.
The Serve-All was tuned to him now. It removed his hat and coat and
put them in the closet. It loosened his tie, patted a sofa cushion to
his shape and brought him a drink.
Perry might just possibly have adjusted to the situation, but the
Serve-All was over anxious.
He liked to sip a drink. But when he lifted the glass to his lips, an
invisible finger of energy pushed helpfully on the bottom.
Perry strangled.
When he recovered, his rage had crystallized in a definite course of
action.
He looked at the Serve-All and he looked at his hands. Not enough.
He needed something much more. His memory of history recalled
such items as an axe and a sledge hammer, but such no longer
existed.
But the plastic table had legs of substantial heft. A low growl rose in
his throat as he grabbed the table and ripped it to pieces.
The dismayed Serve-All scuttled across the room to repair the
damage.
Perry fended it off with his new club and then smashed downward,
again and again, delighting in the screech of crushed metal and the
tinkling death of transistors, vacuum tubes and servos.
At the center was the tiny reactor box, but that was of solid lead and
steel, that, fortunately, was virtually impervious for radiation safety.
But he didn't care. It was also inert and needn't be destroyed.
So Perry was free; as free as an aging husband who has just
dispatched his jaded wife. He sang a little and danced around the
shattered scraps of plastic and wire and metal.
Then he heard the plaintive bleating beep of sound issuing from the
central core of the Serve-All. He bent over it and read engraved
lettering on the steel: "Central Registry No. C187-D69."
Good God! Any idiot would know that every piece of equipment was
centrally registered and carried a built-in signal to summon repair
machinery.
And destruction of mortgaged property was a criminal offense.
So what now?
Escape?
Escape! He must be out of the house when the repair machine
arrived. He must run and keep running, from the law and the Master
Salesman and Serve-All, Inc.
How much time did he have? Not more than a few minutes for the
smooth central machinery to reach across the city to him; machinery
which even now was on its way to rescue a damaged brother.
Perry snatched his coat from the closet and ran to the door.
Food. If he would hide from the methodical meat grinder of society,
he must have food to live. He raced to the kitchen bar.
There was food there, but he didn't know how to get at it. He had
never before needed to do more than dial up portions for a meal, but
he must have food in containers, food that would not spoil while he
conserved his life on its dwindling supply.
He ripped open a locked panel on the wall. There was food. But the
large containers were locked in place. He clawed at the metal, but
only tore his flesh and dripped blood on the immaculate counter top.
The club he had used on the Serve-All! He recovered the plastic
bludgeon and went to work.
Five minutes later he had dislodged two of the large tins. One said
beans; the other said meat.
Beans dripped a trail of juice across the floor as he ran to the door.
He threw it open.
A repair robot scuttled in and knocked him sprawling on the living
room floor.
Perry stared wildly at the mechanical beast. It hummed anxiously,
retrieving bits of wreckage like a mother bird repairing a broken egg.
Mansfield belly-crawled stealthily back toward the door. He might
make it yet. The robot probably wasn't geared for cop duty.
But the door was blocked.
Perry looked up past the knees and the belted paunch to the face. It
was Master Marlboro.
Perry rose wearily to his feet and dropped the tins of food to the
floor.
"All right," he said, "I give up."
"Really, Mr. Mansfield," Marlboro's lips curled with delicate disgust.
"Isn't this a childish way to treat a beautiful machine?"
"What will you do with me?"
The MS didn't answer. He pulled a contract pad out of his pocket and
started writing.
"You mean you're going to sell me another one?"
Marlboro shoved the pad in his hand.
"I'm quite sure you'll sign this one," he said firmly.
Perry read the sales contract:
For standard consideration, this entitles one Perry Mansfield to all
required services and exclusive use of private quarters in Airy Hills
Sanatarium.
Perry signed.
*** END OF THE PROJECT GUTENBERG EBOOK SALES RESISTANCE
***

Updated editions will replace the previous one—the old editions will
be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States copyright in
these works, so the Foundation (and you!) can copy and distribute it
in the United States without permission and without paying
copyright royalties. Special rules, set forth in the General Terms of
Use part of this license, apply to copying and distributing Project
Gutenberg™ electronic works to protect the PROJECT GUTENBERG™
concept and trademark. Project Gutenberg is a registered trademark,
and may not be used if you charge for an eBook, except by following
the terms of the trademark license, including paying royalties for use
of the Project Gutenberg trademark. If you do not charge anything
for copies of this eBook, complying with the trademark license is
very easy. You may use this eBook for nearly any purpose such as
creation of derivative works, reports, performances and research.
Project Gutenberg eBooks may be modified and printed and given
away—you may do practically ANYTHING in the United States with
eBooks not protected by U.S. copyright law. Redistribution is subject
to the trademark license, especially commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE
 PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK

To protect the Project Gutenberg™ mission of promoting the free

distribution of electronic works, by using or distributing this work (or
any other work associated in any way with the phrase “Project
Gutenberg”), you agree to comply with all the terms of the Full
Project Gutenberg™ License available with this file or online at
www.gutenberg.org/license.

Section 1. General Terms of Use and

Redistributing Project Gutenberg™
electronic works
1.A. By reading or using any part of this Project Gutenberg™
electronic work, you indicate that you have read, understand, agree
to and accept all the terms of this license and intellectual property
(trademark/copyright) agreement. If you do not agree to abide by all
the terms of this agreement, you must cease using and return or
destroy all copies of Project Gutenberg™ electronic works in your
possession. If you paid a fee for obtaining a copy of or access to a
Project Gutenberg™ electronic work and you do not agree to be
bound by the terms of this agreement, you may obtain a refund
from the person or entity to whom you paid the fee as set forth in
paragraph 1.E.8.

1.B. “Project Gutenberg” is a registered trademark. It may only be

used on or associated in any way with an electronic work by people
who agree to be bound by the terms of this agreement. There are a
few things that you can do with most Project Gutenberg™ electronic
works even without complying with the full terms of this agreement.
See paragraph 1.C below. There are a lot of things you can do with
Project Gutenberg™ electronic works if you follow the terms of this
agreement and help preserve free future access to Project
Gutenberg™ electronic works. See paragraph 1.E below.
1.C. The Project Gutenberg Literary Archive Foundation (“the
Foundation” or PGLAF), owns a compilation copyright in the
collection of Project Gutenberg™ electronic works. Nearly all the
individual works in the collection are in the public domain in the
United States. If an individual work is unprotected by copyright law
in the United States and you are located in the United States, we do
not claim a right to prevent you from copying, distributing,
performing, displaying or creating derivative works based on the
work as long as all references to Project Gutenberg are removed. Of
course, we hope that you will support the Project Gutenberg™
mission of promoting free access to electronic works by freely
sharing Project Gutenberg™ works in compliance with the terms of
this agreement for keeping the Project Gutenberg™ name associated
with the work. You can easily comply with the terms of this
agreement by keeping this work in the same format with its attached
full Project Gutenberg™ License when you share it without charge
with others.

1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside the
United States, check the laws of your country in addition to the
terms of this agreement before downloading, copying, displaying,
performing, distributing or creating derivative works based on this
work or any other Project Gutenberg™ work. The Foundation makes
no representations concerning the copyright status of any work in
any country other than the United States.

1.E. Unless you have removed all references to Project Gutenberg:

1.E.1. The following sentence, with active links to, or other

immediate access to, the full Project Gutenberg™ License must
appear prominently whenever any copy of a Project Gutenberg™
work (any work on which the phrase “Project Gutenberg” appears,
or with which the phrase “Project Gutenberg” is associated) is
accessed, displayed, performed, viewed, copied or distributed:
 This eBook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
or re-use it under the terms of the Project Gutenberg License
included with this eBook or online at www.gutenberg.org. If you
are not located in the United States, you will have to check the
laws of the country where you are located before using this
eBook.

1.E.2. If an individual Project Gutenberg™ electronic work is derived

from texts not protected by U.S. copyright law (does not contain a
notice indicating that it is posted with permission of the copyright
holder), the work can be copied and distributed to anyone in the
United States without paying any fees or charges. If you are
redistributing or providing access to a work with the phrase “Project
Gutenberg” associated with or appearing on the work, you must
comply either with the requirements of paragraphs 1.E.1 through
1.E.7 or obtain permission for the use of the work and the Project
Gutenberg™ trademark as set forth in paragraphs 1.E.8 or 1.E.9.

1.E.3. If an individual Project Gutenberg™ electronic work is posted

with the permission of the copyright holder, your use and distribution
must comply with both paragraphs 1.E.1 through 1.E.7 and any
additional terms imposed by the copyright holder. Additional terms
will be linked to the Project Gutenberg™ License for all works posted
with the permission of the copyright holder found at the beginning
of this work.

1.E.4. Do not unlink or detach or remove the full Project

Gutenberg™ License terms from this work, or any files containing a
part of this work or any other work associated with Project
Gutenberg™.

1.E.5. Do not copy, display, perform, distribute or redistribute this

electronic work, or any part of this electronic work, without
prominently displaying the sentence set forth in paragraph 1.E.1
with active links or immediate access to the full terms of the Project
Gutenberg™ License.

1.E.6. You may convert to and distribute this work in any binary,
compressed, marked up, nonproprietary or proprietary form,
including any word processing or hypertext form. However, if you
provide access to or distribute copies of a Project Gutenberg™ work
in a format other than “Plain Vanilla ASCII” or other format used in
the official version posted on the official Project Gutenberg™ website
(www.gutenberg.org), you must, at no additional cost, fee or
expense to the user, provide a copy, a means of exporting a copy, or
a means of obtaining a copy upon request, of the work in its original
“Plain Vanilla ASCII” or other form. Any alternate format must
include the full Project Gutenberg™ License as specified in
paragraph 1.E.1.

1.E.7. Do not charge a fee for access to, viewing, displaying,

performing, copying or distributing any Project Gutenberg™ works
unless you comply with paragraph 1.E.8 or 1.E.9.

1.E.8. You may charge a reasonable fee for copies of or providing

access to or distributing Project Gutenberg™ electronic works
provided that:

• You pay a royalty fee of 20% of the gross profits you derive
from the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
 about donations to the Project Gutenberg Literary Archive
Foundation.”

• You provide a full refund of any money paid by a user who

notifies you in writing (or by e-mail) within 30 days of receipt
that s/he does not agree to the terms of the full Project
Gutenberg™ License. You must require such a user to return or
destroy all copies of the works possessed in a physical medium
and discontinue all use of and all access to other copies of
Project Gutenberg™ works.

• You provide, in accordance with paragraph 1.F.3, a full refund of

any money paid for a work or a replacement copy, if a defect in
the electronic work is discovered and reported to you within 90
days of receipt of the work.

• You comply with all other terms of this agreement for free
distribution of Project Gutenberg™ works.

1.E.9. If you wish to charge a fee or distribute a Project Gutenberg™

electronic work or group of works on different terms than are set
forth in this agreement, you must obtain permission in writing from
the Project Gutenberg Literary Archive Foundation, the manager of
the Project Gutenberg™ trademark. Contact the Foundation as set
forth in Section 3 below.

1.F.

1.F.1. Project Gutenberg volunteers and employees expend

considerable effort to identify, do copyright research on, transcribe
and proofread works not protected by U.S. copyright law in creating
the Project Gutenberg™ collection. Despite these efforts, Project
Gutenberg™ electronic works, and the medium on which they may
be stored, may contain “Defects,” such as, but not limited to,
incomplete, inaccurate or corrupt data, transcription errors, a
copyright or other intellectual property infringement, a defective or
damaged disk or other medium, a computer virus, or computer
codes that damage or cannot be read by your equipment.

1.F.2. LIMITED WARRANTY, DISCLAIMER OF DAMAGES - Except for

the “Right of Replacement or Refund” described in paragraph 1.F.3,
the Project Gutenberg Literary Archive Foundation, the owner of the
Project Gutenberg™ trademark, and any other party distributing a
Project Gutenberg™ electronic work under this agreement, disclaim
all liability to you for damages, costs and expenses, including legal
fees. YOU AGREE THAT YOU HAVE NO REMEDIES FOR
NEGLIGENCE, STRICT LIABILITY, BREACH OF WARRANTY OR
BREACH OF CONTRACT EXCEPT THOSE PROVIDED IN PARAGRAPH
1.F.3. YOU AGREE THAT THE FOUNDATION, THE TRADEMARK
OWNER, AND ANY DISTRIBUTOR UNDER THIS AGREEMENT WILL
NOT BE LIABLE TO YOU FOR ACTUAL, DIRECT, INDIRECT,
CONSEQUENTIAL, PUNITIVE OR INCIDENTAL DAMAGES EVEN IF
YOU GIVE NOTICE OF THE POSSIBILITY OF SUCH DAMAGE.

1.F.3. LIMITED RIGHT OF REPLACEMENT OR REFUND - If you

discover a defect in this electronic work within 90 days of receiving
it, you can receive a refund of the money (if any) you paid for it by
sending a written explanation to the person you received the work
from. If you received the work on a physical medium, you must
return the medium with your written explanation. The person or
entity that provided you with the defective work may elect to provide
a replacement copy in lieu of a refund. If you received the work
electronically, the person or entity providing it to you may choose to
give you a second opportunity to receive the work electronically in
lieu of a refund. If the second copy is also defective, you may
demand a refund in writing without further opportunities to fix the
problem.

1.F.4. Except for the limited right of replacement or refund set forth
in paragraph 1.F.3, this work is provided to you ‘AS-IS’, WITH NO
OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED,
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

textbookfull.com