CHAPTER 1 preserves and stains cellular lipids as well as
HISTOLOGY AND ITS METHOD OF STUDY
study of the body tissues and its arrangement to
constitute organs
involves all aspects of tissue biology
how cells structure and arrangements optimize functions
specific to each organ
tissues have two interacting components
cells
ECM (extracellular matrix)
- consists of macromolecules that forms complex
structures
- supports the cells
- contains the fluid transporting nutrients to the cells,
and carrying away their wastes and secretory
products
cells and ECM are strongly influenced by matrix
molecules
- binds to specific cell surface receptors
- connect to structural components inside the cells
organs- formed by an orderly combination of tissues
the precise arrangement of tissues allows the
functioning of each organ and the organism as a
whole
Preparation of Tissues for Study
sections - the most common procedure used in
histologic research
Fixation- preserves tissue structure and prevent
degradation
fixative- solutions of stabilizing or cross-linking
compounds
- formalin- buffered isotonic solution of 37%
formaldehyde
- glutaraldehyde- fixative used for electron
microscopy
cross-links adjacent proteins, reinforcing cell and
ECM structures
Glutaraldehyde treated tissue is then immersed in
buffered osmium tetroxide
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proteins
- Formaline and glutaraldehyde react with the amine
groups (NH2 ), preventing the degradation by
common proteases
tissues are usually cut into small fragments before
fixation to facilitate penetration
- for large organs, fixatives are introduced via blood
vessels
Dehydration- removes water
water is extracted gradually by transfers through a
series of increasing ethanol solution
Clearing- removes alcohol
ethanol is replaced by an organic solvent miscible with
both alcohol and the embedding medium
gives tissue a translucent appearance
Infiltration- tissues is placed in melted paraffin in an
oven at 52- 600 C
evaporates clearing solvent
Embedding
tissues are embedded in a material that imparts firm
consistency
tissue is allowed to harden in a small container of
paraffin at a room temperature
paraffin- for light microscopy
plastic resins- for both light and electron microscopy
- tissues embedded in plastic resin are also
dehydrated in ethanol and infiltrated with plastic
solvents
harden when cross-linking polymerizes are
added
- avoids the higher temperature needed with paraffin
which helps avoid tissue distortions
Sectioning
after embedding, tissue is trimmed
sections are cut at 3-10 m for light microscopy
- electron microscopy requires less than 1 m
microtome- used for sectioning paraffin-embedded
tissues for light microscopy
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 Staining
dyes stain- often behaves like acid or basic
compounds
- forms electrostatic (salt) linkages with ionizable
radicals of macromolecules
basophilic- anionic (net negative charge) like nucleic
acids have an affinity for basic dyes
- e.g. toluidine blue, alcian blue, methylene blue,
hematoxylin
- tissue reacts with basic dyes because of acids in
the tissue
e.g. DNA, RNA, and glycosaminoglycans
acidophilic- cationic components like proteins have
an affinity for acidic dyes
- e.g. eosin, orange G, and acid fuchsin
- stains mitochondria, secretory granules and
collagen
hematoxylin and eosin (H&E)
- most commonly used dye stain
- Hematoxylin - stains DNA, RNA rich portions of
cytoplasm, and the matrix of cartilage
dark blue or purple color
- Eosin - stains other cytoplasmic structures and
collagen
pink
counterstain- dye applied separately to
distinguish additional features
trichome stain- allow greater distinctions
- e.g. Masson trichrome
periodic acid-Schiff (PAS) reaction- utilizes hexose
rings of polysaccharides and other carbohydrate rich
tissue structures
- stains purple or magenta
- Feulgen reaction- stains the DNA of cell nuclei
- PAS- positive material can be identified by enzyme
digestion
pretreatment of a tissue section with an enzyme
that specifically digests one substrate
pretreatment with ribonuclease reduces
cytoplasmic basophilia with little effect on the
nucleus , indicating the importance of RNA for
cytoplasmic staining
Lipid-rich structures
- avoids the processing steps that remove lipids,
such as treatment with heat and organic solvents
- Sudan Black- lipid soluble dye
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useful in diagnosing metabolic diseases that
involve intracellular accumulations of cholesterol,
phospholipids or glycolypids
metal impregnation- uses solution of silver salt to
ECM fibers and cellular elements in the nervous tissue
slide preparation may take from 12 hours to 2 1/2 days
depending on the size of the tissue, the embedding
medium and the method of staining
mounting- final step in slide preparation
Light Microscopy
based on the interaction of light with the tissue
components to reveal and stay features
Bright-Field Microscopy
tissues is examined with ordinary light passing through
the preparation
optical components
- condenser- focuses light
- objective- enlarge and protects the image toward
the observer
- eyepiece (ocular lens)- further magnifies the
image
Total magnification= magnifying power of the
objective x magnifying power of ocular lens
resolving power- smallest distance between 2
structures at which they can be seen as separate
objects
- determines the quality of the image, its clarity and
richness of detail
- maximum resolving power of light microscope - 2
m
Objects smaller than 2 m cannot be seen such
as ribosome or cytoplasmic microfilament
- objective lens with high magnification have high
resolution
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 - ocular lens only enlarges the image and does not differential interference microscopy - produces an
improve resolution image of living cell in 3D
virtual microscopy- used for study of bright-field Confocal Microscopy
microscopic preparations achieves high resolution and sharp focus using
- involves the conversion of stained tissue 1. small point of high-intensity light often from laser
preparation to high-resolution digital images 2. plate with a pinhole aperture in front of the image
- permits study of tissues using a computer without detector
an actual stained slide or microscope unfocused light does not pass through the pinhole
- regions of glass mounted specimen are captured beam splitter- computer driven mirror system moves
digitally in a grid-like pattern at multiple the point of illumination across the specimen
magnifications using specialized slide-scanning automatically and rapidly
microscope -> software converts data for storage Polarizing Microscopy
Fluorescence Microscopy allows the recognition of stained or unstained
fluorescence when certain cellular substances are structures made of highly organized subunits
irradiated by light of proper wavelength, they emit light tissues appear as bright in a dark background
with longer wavelength birefringence- ability to rotate the direction of
tissues sections are usually irradiated with UV light vibration of polarized light
and the emission is in the visible portion of the - feature of crystalline substances containing highly
spectrum oriented molecules
- fluorescent substances appear bright on a dark e.g. cellulose, collagen, microtubules and actin
background filaments
fluorescent stains- fluorescent compounds with Bright field uses ordinary light
affinity for specific cell macromolecules Microscopy
- Acridine orange- binds both DNA and RNA
Nucleic acids emit slightly different fluorescence,
allowing them to be localized separately in cells
- DAPI and Hoechst stain- specifically bind DNA Fluorescence uses ultraviolet light
Microscopy
and are used to stain cell nuclei
emits a characteristic blue fluorescence under UV
- coupling fluorescein to molecules that will
Phase-Contrast uses difference in
specifically bind to cellular components allows the
Microscopy refractive indices
identification of these structures under the
microscope
- antibodies labeled with fluorescent compounds are
Confocal uses focal planes
extremely important in immunohistologic staining Microscopy and light beam
Phase-Contrast Microscopy - uses lens system that
produces visible images from transparent objects
uses the difference in refractive inde
can be used with living, cultured cells
based on the principle that light changes its speed Electron Microscopy
when passing through cellular and extracellular
structures with different refractive indices based on the interaction of tissue components with
causes the structures to appear lighter or darker in beams of electrons
relation to each other 1000-fold increase in resolution
allows the examination of cells without fixing or Transmission Electron Microscopy
staining permits resolution around 3 nm
beam of electrons focused using electromagnetic
lenses passes through the tissue section
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 produces an image with black, white and intermediate
shades of gray regions
- brighter and electron lucent- electrons pass
readily
- darker and electron dense- electrons were
absorbed or deflected
compounds with heavy metal ions are often added to
fixative or dehydrating solutions
- e,g. osmium tetroxide, lead citrate, uranyl
compounds
cryofracture and freeze etching- allow TEM study of
cells without fixation or embedding
- used in the study of membrane structures
- specimens are frozen in liquid nitrogen and cut with
a knife
- thin coats of platinum is applied
Scanning Electron Microscope
provides high resolution view of the surfaces of cells,
tissues and organs
beam does not pass through the specimen
surface is coated with layer of heavy metal (gold)
which reflects electrons
- reflected electrons are captured by a detector
presents a three-dimensional view
Autoradiography
method of localizing newly synthesized macromolecules
slides with radio labeled cells or tissue sections are
coated in a darkroom with photographic emulsions
silver bromide crystals- act as microdetectors of the
radiation
- produced small black grains of metallic silver, which
indicates the locations of radio labeled molecules in
the tissue
radioactive precursor of DNA (tritium-labeled
thymidine)- knows which cells in the tissue are replicating
DNA
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Cell and Tissue Culture
In vitro- live cells are studied outside the body in culture
in vivo- in the organism
cells are bathed in fluid derived from blood plasma
cell culture- allows the direct observation of cells
behavior under a phase contrast microscope
primary cell culture- cells to be cultured are
dispersed and placed in a clear dish to which they
adhere in single layers
cells can be maintained in vitro for a long period of time
because they become immortalized and constitute a
permanent cell line
a cell culture developed from a single cell and
therefore consisting of cells with a uniform genetic
makeup
HeLa cells
- Cervical cancer cells from Henrietta Lacks
- still used in research on cellular structure until now
transformation- promote cell immortality
Enzyme Histochemistry
method for localizing cellular structures using a specific
enzymatic activity
usually use unfixed and mildly fixed tissue which is
sectioned in a cryostat
for histochemistry,
tissue sections are immersed in a solution containing
the substrate of the enzyme to be localized
enzyme is allowed to act on its substrate
section is then put in contact with a marker compound
that reacts with the product of the enzymatic action on
the substrate
the product which is insoluble and visible by
microscopy precipitates over the site of the enzyme,
identifying their location
enzymes that can be detected in histochemistry
phosphatases- remove phosphate groups from
macromolecules
dehydrogenase- transfer hydrogen ions from one
substrate to another
peroxidase- promotes the oxidation of substrates with
the transfer of hydrogen ions to hydrogen peroxide
histochemical procedure used in the medical laboratory
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 Perls Prussian blue reaction for iron- diagnose iron
storage diseases hemochromatosis and
hemosiderosis
PAS-Amylase and Alcian blue reactions for
polysaccharide- detect glycogenesis and
mucopolysachharidosis
reactions for lipids- detect sphingolipidosis
Visualizing Specific Molecules
most commonly used labels are fluorescent compounds,
radioactive atoms, molecules of peroxidase and metal
particles that can be seen with microscopy
molecules that interact specifically with other molecules
Phalloidin- interacts strongly with actin proteins of
microfilaments
- extracted from mushroom Amanita phalloides
Protein A- binds to Fc region of antibody molecules
- from Staphylococcus aureus
Lectins- bind to carbohydrate with high affinity and
specificity
- derived from plant seed seeds
Immunohistochemistry
identifies and localizes many specific proteins
antibodies- against antigens that are recognized as
foreign
- belong to immunoglobulin family of glycoproteins
- secreted by lymphocytes
- to produce antibodies against protein, protein is
injected into an animal of other species
growth of the cell activity can be prolonged by
fusing them with lymphocytic tumor cells to
produce hybridoma cells
- polyclonal antibodies - capable of binding a
different region of protein x
collection of antibodies from different B cells that
recognize multiple epitopes on the same antigen
the part of an antigen molecule to which an
antibody attaches itself
- monoclonal antibodies - represents antibody from
a single antibody producing B cell and therefore
only binds with one unique epitope
it can be selected to be highly specific and to bind
strongly to the protein to be detected
direct immunohistochemistry - if the cell or tussle is
detected by directly binding a labeled primary antibody
specific for that antigen
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indirect immunihistochemistry- uses an unlabeled
primary antibody that is detected bound to its antigen
with labeIed secondary antibodies
- used more widely in research because it is more
sensitive
Hybridization Techniques
implies the specific binding between 2 single strands
of nucleic acids
allows the specific identification of sequences in genes
or RNA
In situ hybridization- when nucleic acid sequences
are applied directly to prepared cells and tissue
sections
this technique is ideal for:
- determining if a cells has a specific sequence of
DNA
- identifying the cells containing specific messenger
RNA
- determining the localization of gene in a specific
choromosome
probe- detects nucleotide sequences
- may be obtained by cloning, polymer chain
reaction, amplification of the target sequence or by
chemical synthesis
Interpretation of Structures in Tissue Sections
artifacts- minor structural abnormalities
minor shrinkage of cells or tissues
- can create artificial spaces between cells and other
tissue components
small wrinkles in the section
precipitates from the stain
when a structures 3d volume is cut in to thin sections,
the section appears 2D only
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