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Intro To Histology and Micros

This document provides an overview of histology, including how tissues are prepared for microscopic examination through fixation, processing, embedding, sectioning, staining, and microscopic analysis. Key steps include fixation in formalin, dehydration in alcohol baths, clearing in xylene, infiltration with paraffin wax, sectioning thin slices on a microtome, staining typically with hematoxylin and eosin, and examining under a light microscope. Special techniques like immunohistochemistry and virtual microscopy are also discussed.

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35 views21 pages

Intro To Histology and Micros

This document provides an overview of histology, including how tissues are prepared for microscopic examination through fixation, processing, embedding, sectioning, staining, and microscopic analysis. Key steps include fixation in formalin, dehydration in alcohol baths, clearing in xylene, infiltration with paraffin wax, sectioning thin slices on a microtome, staining typically with hematoxylin and eosin, and examining under a light microscope. Special techniques like immunohistochemistry and virtual microscopy are also discussed.

Uploaded by

rebeccaboukenj
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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INTRODUCTION TO

HISTOLOGY:
How to make visible the invisible
Reference  Wheater’s functional histology

book
 Study of the microscopic anatomy of cells and tissues

What is  Performed by examining a thin slice (section) of tissue under a


light microscope (tissues are too thick for light to pass through
Histology? them)
 Use of histological stains (Hematoxylin&Eosin)
 Histologic examination requires fixation of the tissues. Fixation
preserves tissues and prevent autolysis and degradation.
 Most common fixative is 10% neutral buffered formalin
 The tissue is immersed in a volume of fixative that is at least 5x its
Fixative Agent volume.
 Big tissues samples should be dissected and left in formalin
overnight to allow the fixative agent to penetrate the tissue and
avoid deterioration
Tissues are placed in
cassettes for processing
Tissue
processing
Cassettes will be processed overnight in the processing machine in
several steps:
-Formalin, for additional fixation
-Alcohol, successive immersion in alcohol bathe starting with 70%
to 80% to 95% to 100% for tissue dehydration
Processing -Xylene, for tissue clearing to remove alcohol
-Paraffin, (a kind of wax) that will infiltrate the tissue and preserve it
and makes it suitable for microtomy
Note that this process takes around 13hrs and usually takes place
overnight
Our purpose is to have a
paraffin block so we can  Embedding
cut thin slices on the  Sectioning
microtome, for that, will
have to follow several  Staining
steps:  Cover slipping and labeling
Purpose is to embed in a solid medium
to preserve and facilitate sectioning
 Tissue samples are placed into
molds, liquid paraffin is added and
will be hardened by cooling
 The hardened blocks containing
Embedding the tissue samples are ready to be
sectioned
 Formalin-fixed, paraffin-embedded
(FFPE) tissues may be stored
indefinitely at room temperature
(archives)
 Nucleic acids (both DNA and RNA)
may be recovered from them
decades after fixation, making
FFPE tissues an important resource
for historical studies in medicine
Embedding
(video)
Purpose is to cut by microtome thin sections that we can stain and
Sectioning perform additional studies on

(microtomy)  Sections are cut at 3-4µm in thickness and placed on a glass slide
Sectioning,
Trimming
(video)
Sectioning,
cutting (video)
Purpose is to highlight particular features of interest
 Recommended stain is Hematoxylin and eosin, where
hematoxylin stains nucleus blue while eosin stains
cytoplasm pink
Staining  Staining can be done manually or automated
Purpose is to preserve the tissue so that sections won’t be scratched

Cover slipping In pathology, Paraffin blocks and slides should be archived in the
department for at least 7-10 years so they can be retrieved if needed
and labeling by the patient.
 Special staining: selectively stain cells and cellular components
(Trichrome, Periodic Acid Schiff PAS, Alcian Blue, Perls…)

 Immunohistochemistry (IHC): identify categories of cells.


Antibodies employed to specifically visualize specific antigens in a
Special tissue. Antibody must be prelinked to an indicator substance =
enzyme that is able to convert a colorless substrate to a brown
techniques colored product

 Immunofluorescence (fluorescent microscope): uses a


fluorescent molecule (fluorescein) to label antibodies targeting a
specific molecule.
Special
stains
Immunohisto-
chemistry
 Other advanced techniques, such as in situ hybridization, are
available to identify specific DNA or RNA molecules present in the
tissue
 Fluorescent probes can be used
 Fluorescence microscopy with special filters is used to detect
fluorescent signals
Special
techniques
 Always start with low magnification 2.5 or 5x and go gradually to
high magnification, 10x, 20x and 40x

Microscopic
 To read on 100x we should use oil
examination
 Always scan the entire slide when looking for abnormal cells
Aperio E-slide manager website
It’s our virtual/digital slides website that you can access from
virtual-microscope.lau.edu.lb
 Username is your LAU email address

Virtual  Password is 123456, the system will ask you to change it when you
first log in.
microscope
Note: It is preferable that you access the link from LAU, since the slides need
high speed internet to download.
Welcome to our school 

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