Animal Histology Lecture 1

Introduction of Animal
Methods used in Histology
Histology is the study of tissue. To examine the tissue components,
it is necessary to process the tissue. The steps of processing
involve: fixation, sectioning, and visualization.
Making histological slide
1. Making histological slide
a. Fixation--prevent decomposition, cell metabolism is stopped
b. Dehydration—For LM and TEM samples
b. Embedding– LM using paraffin, TEM using plastic monomers
c. Sectioning—LM 5-10µm, TEM using 0.02-1.0µm thick section
d. Staining—LM sections with dyes or fluorescent tags, TEM treating the
section with heavy metal salts
e. Cover slipping
• The branch of science that deals with the
chemical composition of the cells and
tissues of the body , including cytochemistry
• Immunohistochemistry is a method of
analyzing and identifying cell types
based on the binding of antibodies to
specific components of the cell. It is
sometimes referred to as
4. Microautoradiography
• Technique for the study of surfaces of
solids by monochromatic-radiation (such
as x-ray) contrast effects shown via
projection or enlargement of a contact
B. Vital(live) tissue study
1.Tissue culture(cell preparation)
2.Vital staining(Isotope)
A technique in which a harmless dye is used to stain living tissue for microscopical observation. The stain may be
injected into a living animal and the stained tissue removed and examined (intravital staining) or the living tissue
may be removed directly and subsequently stained (supravital staining). Microscopic organisms, such as protozoa,
may be completely immersed in the dye solution. Vital stains include trypan blue, vital red, and Janus green, the
latter being especially suitable for observing mitochondria.
Read more: vital staining - intravital staining, supravital staining - Living, Tissue, and Dye
3.Cell fusion(cloning)
4. Cell electrophoresis
C. New technology used in Histology study
• Freeze-fracture-etch
overcomes the inherent limitation of thin specimen sections to real details of the internal
structure of membranes and intercellular junctions.
• Cell morphometry
• Flow cytometry(FCM)
• Microspectrophotometry
D. Microscope
1.Light microscope(LM)
a. Biomicroscope
b. Fluorescence microscope
c. Phase contrast microscope
d. Darkfield microscope
e. Polarizing microscope
f. Confocal laser scanning microscope
2.Electron microscope, EM
• Transmission electron microscope (TEM)
• Scanning electron microscope(SEM)
• Ultrahigh voltage electron microscope
Staining reaction
Acidophila-tissue components that binds acidic dyes(eosin, orange C), +
Erythrocyte cytoplasm, collagen fibers, mitochondria, lysosomes
Basophila- tissue binds basic dyes(hematoxylin, methylene blue), Nuclei,RER,extracellular matrix
• Metachromasia-change in color, is a property of certain dyes(usually
basic dyes,(low-blue), (high-purple)
• Hematoxylin-Eosin (HE) Reaction
• Periodic Acid Schiff Reaction (PAS)
This an all-around useful stain for many things. It stains glycogen,
mucin, mucoprotein, glycoprotein, as well as fungi. A predigestion
step with amylase will remove staining for glycogen. PAS is useful
for outlining tissue structures--basement membranes, capsules,
blood vessels, etc. It does stain a lot of things and, therefore, can
have a high background. It is very sensitive, but specificity depends
upon interpretation.
• Silver staining
Hematoxylin and Eosin:
The most common stain
used in histology is
hematoxylin and eosin or
H&E. Hematoxylin
stains acidic tissue
components such as the
RNA in these nuclei
purple. Acidic tissue
components stained with
this basic dye are
referred to as basophilic
structures. Eosin stains
components pink to
orange; components
which react with this
acidic dye are acidophilic.
H and E staining
Hematoxylin is the oxidized product of the logwood tree known as hematein. Since
this tree is very rare nowadays, most hematein is of the synthetic variety. In order to
use it as a stain it must be "ripened" or oxidized. This can be done naturally by
putting the hematein solution on the shelf and waiting several months, or by buying
commercially ripened hematoxylin or by putting ripening agents in the hematein
Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the
tissues. This is provided by a metal cation such as iron, aluminum, or tungsten. The
variety of hematoxylins available for use is based partially on choice of metal ion
used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity
for the nucleic acids of the cell nucleus.
Hematoxylin stains are either "regressive" or "progressive". With a regressive stain,
the slides are left in the solution for a set period of time and then taken back through
a solution such as acid-alcohol that removes part of the stain. This method works
best for large batches of slides to be stained and is more predictable on a day to
day basis. With a progressive stain the slide is dipped in the hematoxylin until the
desired intensity of staining is achieved, such as with a frozen section. This is
simple for a single slide, but lends itself poorly to batch processing.
Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. There
are a variety of eosins that can be synthesized for use, varying in their hue, but they
all work about the same. Eosin is much more forgiving than hematoxylin and is less
of a problem in the lab. About the only problem you will see is overstaining,
especially with decalcified tissues.
Microscopy The microscope is an important tool in your study of histology. You need to understand and
have a working knowledge of the Parts of the microscope and what objective engravings mean so you
can use this tool most efficiently. Also, the principles of image formation are important. As you work through
your slide set, you will be using slides stained with hematoxylin and eosin plus a variety of special stains
that reveal different structures within the tissue. In addition, you must be able to recognize artifacts
of preparation.
Parts of the microscope
The image on the left indicates
the major parts of the microscope.
Objective engravings
The engravings on the microscope objective provide information about
the use of the objective. The objective below is a flat field objective
which means the image is in focus across the entire field.
Magnification is the number of times the image of the original specimen
is increased in size by the objective lens. The numerical aperture
is related to resolution; the higher the N.A., the higher the resolving power of
the objective. Tube length is the distance from the objective lens to the eyepiece
objective. Objectives should be used with a microscope body having matched tube
length. Coverslip thickness indicates the thickness of the glass which should be
used to cover the specimen.
Image formation through the
Scroll to the bottom of this
diagram and note that the light
source emits light which travels
through the condenser lens and
then the specimen. The objective
lens projects an inverted and
reverted primary image. The
eyepiece lens, in concert with the
lens of the eye, forms a
secondary image which is again
inverted and reverted (or normal
in orientation). The brain
interprets the secondary image
and projects it back to the level
of the stage as a virtual image
which is once again inverted and
reverted. Thus, the image that
you view through the microscope
is "upside down and backwards."
Special stains:
Special stains, other than the standard hematoxylin and eosin (H&E) stain, can
give you more information about the tissue. To the left is a PAS stain which
emphasizes the basement membrane (black arrow) and the brush border (blue
arrow) of the cells. The stain colors carbohydrates in these regions. These
structures would not be as heavily stained with H&E.
This trichrome stain helps differentiate muscle fibers (red) from
connective tissue proper (blue). With H&E, both of these tissue
would stain pink with eosin.
Gridley's reticulum stain emphasizes fine black
reticular fibers which would not stand out in a
standard H&E preparation.
Artifacts in tissue preparations:
The processing of tissues sometimes results in artifacts in the final tissue
section. Some more common artifacts are shown in the image to the left. (1)
Extraneous fiber on the coverslip. Note how it is out of focus. (2) Tissue fold the tissue has folded on top of itself. (3) Chatter - cracks in the tissue section
which occur during cutting.
The images on the left show the tissue and debris (blue arrow) in focus on the far
left. The fiber (black arrows) is out of focus and thus, above the plane of the
tissue. When the fiber is in focus (right image), the plane of the tissue is out of
focus. You can use this focusing principle to determine if a structure is within the
tissue or not.
Basic Cell Biology
The cell is the basic structural unit of the tissue and
organs of the body. Various Cell shapes include spherical,
stellate, spindle, polyhedral, squamous, cubodial
Or columnar. Cell sizes range from a single micrometer
to several centimeters in diameter
Cell Membrane (1)
• Each cell in the body is bounded by a cell
membrane (plasma lemma) which provides a
barrier and controls movement of substances
into and out of the cell. The cell membrane is a
lipid bilayer with embedded proteins.
• Integral proteins are tightly bound within the
membrane and often extend across it as
transmembrane proteins. These transmembrane
proteinss frequently form ion channels or carrier
proteins that transport molecules across the cell
Cell Membrane (2)
• Peripheral proteins, located on the
cytoplasmic surface of the cell membrane,
are more loosely bound to other
membrane proteins or lipids.
• A glycocalyx, comprised of carbohydrates
on the outer surface of the cell membrane,
functions in cell recognition, adhesion,
absorption and antigenicity
• More than one nucleus can be present in a cell.
Within this spherical structure, deoxyribonucleic
acid (DNA) is transcribed and ribonucleic acid
(RNA) is synthesized. The surrounding nuclear
envelope is formed by two adjacent bilaminar
lipid membranes with embedded proteins.
Scattered nuclear pores, which perforate the
envelope, regulate passage of substances
between the cytoplasm and the nucleus.
• Chromatin, primarily comprised of DNA, is
located within the nucleus. The inert form of
chromatin, heterochromatin, stains intensely
while euchromatin, which is actively involved in
protein production, stains lightly. Nuclear
chromatin condenses to form chromosomes
during cell division. Also within the nucleus is the
nucleus is the nucleolus that is the site of rRNA
synthesis. The number and size of cell nucleoli
are related to the amount of protein synthesis
occurring within the cell.
• The cytoplasm, which surrounds the
nucleus and organelles of the cell, varies
in composition of water, protein,
carbohydrates and salts.
• A cytoskeleton of microfilaments,
intermediate filaments and microtubles
provides structure for cell shape and
Organelles and inclusions (1)
• Granular endoplasmic reticulum (rough
endoplasmic reticulum, rER) is comprised of
membranous cisternae with attached ribosomes.
The ribosomes, made up of ribosomal RNA,
translate messenger RNA from nucleus. As a
result of the translation, specific proteins are
formed within the lumen of the cisternae and are
subsequently packaged for export outside the
cell. Free ribosomes may also be present in the
cytoplasm and function in the production of
intracellular proteins.
Organelles and inclusions (2)
• Agranular endoplasmic reticulum (smooth
endoplasmic reticulum, sER) lacks ribosomes
and is associated with glycogen synthesis,
steroid production and cell detoxification.
• The Golgi comples, a curved stack of
membranes in the cytoplasm, receives proteincontaining transport vesicles from the rER on the
convex surface and releases secretory vesicles
from the concave surface. While passing
through the Golgi complex, proteins are
glycosylated, phosphorylated or sulfated in
preparation for export.
Organelles and inclusions (3)
• Mitochondria have a smooth outer
membrane while the inner membrane is
folded into characteristic cristae. Matrix
granules are also present. Mitochondria
produce chemical energy for the cell
through the tricarboxylic acid cycle,
oxidative phosphorylation and fatty acid
• Many different types of vesicles containing
material being transprted through the cell are
also presnet in the cytoplasm. These vesicles
may be coated with materials such as clathrin or
coatomer. The vesicles shuttle between the cell
surface, Golgi apparatus, granular endoplasmic
reticulum or lysosomes depending on the
transport pathway.
• Other inclusions within the cell often include
secretory granules, nutrients such as glycogen
and lipid, and pigments.
Cell Surface Modifications
• Depending on cell shape and function, the surface
membrane may be modified to form special stuctures.
Microvilli are short, finger-like projections of the cell
membrane which are non-motile, but dramatically
increase cell surface area for absorptive functions.
• Stereocilia are long microvilli limited to the epididymis
and ear.
• Cilia and longer flagella contain microtubules arranged
in a characteristic pattern. Nine pairs of microtubules
occupy the periphery of the cilium or flagellum while one
pair is found in the center. Both cilia and flagella are
motile. Another cilium-like structure, the kinocilium, is
found in the ear.
The cell---Overview
• The cell is the basic structural unit of tissues
• A lipid bilayer with embedded proteins forms the cell
• The nucleus controls cell function
• Granular endoplasmic reticulum and associated
ribosomes produce proteins
• Agranular endoplasmic reticulum is assocaited with
steroid production and detoxification
• The Golgi complex packages protein for export
• Mitochondria provide energy for the cell
• Cell surface modifications include microvilli, cilia, flagella
and stereocilia.
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