HISTOLOGY

LESSON 1: INTRODUCTION A. FIXATION

 Preserving fresh tissue for examination.
I. DEFINITON OF HISTOLOGY  Fixatives have the property of forming
II. PREPARATION OF TISSUE FOR cross-links between proteins.
STUDY  Widely used fixative for light
microscopy is formalin, a buffered
A. Fixation isotonic solution of 37% formaldehyde.
B. Decalcification
C. Dehydration  Primary Aim: To preserve the
D. Clearing morphologic and chemical integrity of
E. Infiltration the cell as life-like manner as possible.
F. Embedding  Secondary Aim: To harden and protect
G. Sectioning the tissue from the trauma of further
H. Staining handling.

III. LIGHT MICROSCOPY Practical considerations of Fixation:

 speed
A. Bright-field Microscopy (definition,  duration of fixation
parts, and functions)
 penetration (1mm/hr)
B. Fluorescence Microscopy
 volume of fixative (fixative-to-tissue
C. Phase-contrast Microscopy
ratio 20:1)
D. Confocal Microscopy
E. Polarizing Microscopy
B. DECALCIFICATION
IV. ELECTRON MICROSCOPY  More concentrated acid solutions
decalcify bone more rapidly but are
A. Transmission Electron Microscopy more harmful to the tissues.
B. Scanning Microscopy  High concentrations and greater amount
of fluid will increase the speed of the
process.
I. HISTOLOGY  Dense bone tissues usually require up to
14 days or longer in order to complete
 Study of the tissues. the process.
 The Greek root histo can be translated as
either "tissue" or "web". C. DEHYDRATION
 An aspect of tissue biology with the  to remove the fixative and water from
focus on how cell structure and the tissues and replacing them with
arrangement optimize functions specific dehydrating fluid
to each organs − advances in molecular  generally used in increasing strengths
biology, physiology, immunology, and (all the aqueous tissue fluids are
pathology are essential for a better removed but with the little disruption to
knowledge of tissue biology. the tissue due to diffusion currents)

II. PREPARATION OF TISSUE • Ethanol — for routine dehydration of tissues.

FOR STUDY • Methyl alcohol — employed for blood and
tissue films.
The most common procedure used in • Butyl alcohol — for plant and animal micro
histologic research is the preparation of tissue techniques.
slices or “sections” that can be examined
visually with transmitted light. D. CLEARING

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  Removal of alcohol. deparaffinized, and stained for light microscope
 Should be miscible with BOTH the study.
dehydrating fluid and the embedding
medium. H. STANING
 Clearing agent gives the tissue a  Most cells and extracellular materials
translucent appearance. are completely, and to be studied
microscopically the tissue sections must
• Xylene — most common and most rapid be stained.
clearing agent.  Cell components such as nucleic acids
with a negative charge (anionic) have an
E. INFILTRATION affinity for basic dyes and are termed
 The tissue is then placed in melted basophilic.
paraffin until it becomes completely  Examples of Basic dyes:
infiltrated with this substance.  Toluidine blue
 Alcian Blue
• Paraffin — simplest/most common -not  Methylene Blue
recommended for fatty tissues.
• 55-60ºC — Temperature of paraffin oven  glycosaminoglycan, acid
(paraffin oven must be maintained at a glycoprotein, nucleic acids
temperature 2-5ºC above the melting point of the (reacts with basic dyes because
paraffin wax). of the acids in their
composition)
F. EMBEDDING  Cationic components such as proteins
 Infiltrated tissue is placed into a stain more readily with acidic dyes and
precisely arranged position in a mold are termed acidophilic.
containing a medium which is then  Examples of Acid dyes:
allowed to solidify. — Eosin
— Orange G
G. SECTIONING — Acid Fuchsin
 Paraffin block is cut into uniformly thin
slices to facilitate studies under the — Cell components that are
microscope. acidophilic are mitochondria, secretory
granules, and collagen.
 3-10um = for routine histologic sections
 10-15um = frozen section
 less than 1um = electron microscopy Other examples of stains:

 Hematoxylin — stains DNA of the cell

MICROTOME — is used for sectioning nucleus and other acidic structures such
paraffin embedded tissues for light microscopy. as RNA-rich proteins of the cytoplasm
The trimmed tissue specimen is mounted in the and matrix of the cartilage producing a
paraffin block holder, and each turn of the drive DARK BLUE or PURPLE color.
wheel by the histologist advances the holder a
controlled distance, generally from 1 to 10 μm.  Eosin — stains other cytoplasmic
After each forward move, the tissue block passes structure and collagen PINK -acts as
over the steel knife edge and a section is cut at a counterstain in H&E stain.
thickness equal to the distance the block
advanced. The paraffin sections are placed on  Periodic Acid Schiff — utilizes the
glass slides and allowed to adhere, hexose rings of polysaccharides and
other carbohydrate-rich tissue structures

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 and stains such macromolecules toward the eyepiece. Interchangeable
distinctly PURPLE or MAGENTA. objectives with different magnifications
routinely used in histology include X4
 Sudan Black — a lipid soluble dye, for observing a large area (field) of the
useful in diagnosis of metabolic diseases tissue at low magnification; X10 for
that involve intracellular accumulations medium magnification of a smaller
of cholesterol, phospholipids or field; and X40 for high magnification of
glycolipids. more detailed areas.

III. LIGHT MICROSCOPY Diaphragm — regulates the amount of

light on the specimen.
Conventional bright-field microscopy, as well as
more specialized applications like fluorescence, Coarse adjustment knob — moves the
phase-contrast, confocal, and polarizing stage up and down. Used to find a
microscopy, are all based on the interaction of specimen when using the low power
light with tissue components and are used to objective.
reveal and study tissue features.
Fine adjustment knob — moves the
A. Bright-field Microscopy — stained tissue is body tube for focusing the high power
examined with ordinary light passing through lens.
the preparation.
Arm — used to support the microscope
• Resolving Power — defined as the when carried.
smallest distance between two structures at
which they can be seen as separate objects. Stage — the platform that is flat used
• Virtual Microscopy — used for study for placing the slides under observation.
of bright-field microscopic preparations,
involves the conversion of a stained tissue Stage clip — Stage clips hold the slides
preparation to high-resolution digital images and in proper place.
permits study of tissues using a computer or Condenser — collects and focuses a
other digital device, without an actual stained cone of light that illuminates the tissue
slide or a microscope. slide on the stage.

PARTS AND FUNCTIONS OF A BRIGHT – Base — supports the microscope.

FIELD MICROSCOPE
Power switch — turns the light on and
Eyepiece or ocular lens — magnify off.
this image another X10 and project it to
the viewer, yielding a total B. Fluorescence Microscopy — uses ultraviolet
magnification of X40, X100, or X400. light, under which only fluorescent molecules
are visible, allowing localization of fluorescent
Tube — connects the eyepiece to the probes which can be much more specific than
objective lenses. routine stains.

Revolving nosepiece — also known as • Fluorescence — when certain cellular

the Turret. Revolving nosepiece has substances are irradiated by light of a proper
holders for the different objective wavelength, they emit light with a longer
lenses. wavelength.

Objective lenses — enlarge and project • Acridine orange – a fluorescent stain

the illuminated image of the object which binds both DNA and RNA.

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C. Phase-contrast Microscopy  Primary cell cultures — preparations
— It uses the differences in refractive in which cells to be cultured are
index of various natural cell and tissue dispersed mechanically or enzymatically
components to produce an image without from a tissue or organ and placed with
staining, allowing observation of living cells. sterile procedures in a clear dish to
— Based on the principle that light which they adhere, usually as a single
changes its speed when passing through cellular layer.
and extracellular structures with different
refractive indices.  Some cells can be maintained in vitro
for long periods because they become
D. Confocal Microscopy — involves scanning immortalized and constitute a permanent
the specimen at successive focal planes with a cell line.
focused light beam, often from a laser, and
produces a 3D reconstruction from the images.
 Transformation
E. Polarizing Microcopy  The genetic alteration of a cell
— allows the recognition of stained or resulting from the direct uptake and
unstained structures made of highly organized incorporation of exogenous genetic
subunits material from its surroundings
— produces an image only of material through the cell membrane.
having repetitive, periodic macromolecular  Alteration on genetically
structure; features without such structure are not programmed life span of cells that
seen. promote cell immortality are similar
to the initial changes in a normal
• Birefringence — ability to rotate the cell’s becoming a cancer.
direction of vibration of polarized light

AUTORADIOGRAPHY ENZYME HISTOCHEMISTRY

MICROSCOPIC AUTORADIOGRAPHY ENZYME HISTOCHEMISTRY — is

 Is a method of localizing newly a method for localizing cellular
synthesized macromolecules in cells or structures using a specific enzymatic
tissue sections. activity present in those structures.
 Radioactively labeled metabolites
(nucleotides, amino acids, sugars) Pointers to remember for enzyme
provided to the living cells are histochemistry:
incorporated into specific
macromolecules (DNA, RNA, protein, (1) Tissue sections are immersed in a
glycoproteins, and polysaccharides) and solution containing the substrate, a
emit weak radiation that is restricted to substance which an enzyme acts, of the
those regions where the molecules are enzyme to be localized;
located. (2) The enzyme is allowed to act on its
substrate;
CELL AND TISSUE CULTURE (3) The section is then put in contact
with a marker compound that reacts
Cell culture — allows the direct observation of with a product of the enzymatic action
cellular behavior under a phase-contrast on the substrate;
microscope and many experiments technically (4) The final product from the marker,
impossible to perform in the intact animal can be which must be insoluble and visible by
accomplished in vitro. light or electron microscopy,

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 precipitates over the site of the enzymes, IMMUNOHISTOCHEMISTRY
identifying their location.
IMMUNOHISTOCHEMISTRY
Examples of enzymes that can be — Labelled antibodies are routinely
detected histochemically include the used to identify and localize many specific
following: proteins, not just those with enzymatic activity
that can be demonstrated by histochemistry
 Phosphatases that remove phosphate — Based on specific reactions between
groups from macromolecules. antigen and antibodies labeled with visible
 Dehydrogenases, which transfer markers, often fluorescent compounds or
hydrogen ions from one substrate to peroxidase for light microscopy and gold
another, such as many enzymes of the particles for TEM.
citric acid (Krebs) cycle, allowing
histochemical identification of such  Direct immunohistochemistry —
enzymes in mitochondria. process by which the cell or tissue
 Peroxidase, which promotes the antigen of interest is detected by directly
oxidation of substrates with the transfer binding a labelled primary antibody
of hydrogen ions to hydrogen peroxide. specific for that antigen
 Indirect immunohistochemistry —
VISUALIZING SPECIFIC MOLECULES uses an unlabeled primary antibody that
is detected bound to its antigen with
VISUALIZING SPECIFIC MOLECULES — labelled secondary antibodies.
a specific macromolecule present in a tissue
section may also be identified by using tagged The indirect immunohistochemical
compounds or macromolecules that bind method is more commonly used because
specifically with the molecule of interest. the added level of antibody binding
amplifies the signal detected and
Examples of molecules that interact provides greater technical flexibility.
specifically with other molecules include the
following:  In Situ Hybridization (ISH) —
technique in which specific gene
 Phalloidin, a compound extracted from sequences or mRNAs of cells can be
mushroom, Amanita phalloides, detected microscopically using labeled
interacts strongly with the actin protein complementary DNA (cDNA) probes.
of microfilaments.
 Protein A, purified from INTERPRETATION OF STRUCTURES
Staphylococcus aureus bacteria, binds to IN TISSUE SECTIONS
the Fc region of antibody molecules, and
can therefore be used to localize  Many steps in tissue processing, slide
naturally occurring or applied antibodies preparation, and staining can introduce
bound to cell structures. minor artifacts such as spaces and
 Lectins, glycoproteins derived mainly precipitates that are not normally present
from plant seeds, bind to carbohydrates in the living tissue and must be
with high affinity and specificity. recognized.
Different lectins bind to specific sugars
or sequences of sugar residues, allowing  Sections of cells or tissues are
fluorescently labeled lectins to be used essentially 2D planes through 3D
to stain specific glycoproteins or other structures, and understanding this fact is
macromolecules bearing specific important for their correct interpretation
sequences of sugar residues. and study.

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