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Chapter 1

Anatomy and Cell Biology 3309 Chapter Notes - Chapter 1: Glycerol, Osmium, Digoxigenin


Department
Anatomy and Cell Biology
Course Code
ANAT 3309
Professor
Kem Rogers
Chapter
1

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Notes on Chapter 1
Tissue Preparation
Can use light microscopes or electron microscopes to visualize images
EM has higher resolution than LM
Histological slide is 2D, must be able to understand how the image relates to the 3D
structure that it came from
Preparing tissue/organ sample:
1. fixation: usually by chemical/mix of chemicals (eg. Formalin/formaldehyde) to
permanently preserve tissue structure for subsequent treatments; done immediately
after removing organ/tissue; ends cell metabolism, prevents autolysis of cells/tissues;
kills pathogens; hardens tissue by cross-linking or denaturing protein molecules
2. embedding in paraffin: allows tissue to be thinly sliced; washed and dehydrated with
alcohol solutions; alcohol is cleared with xylol or toluol; infiltrate specimen with melted
paraffin; paraffin cools/hardens then is trimmed and sliced
3. mounting: slices stuck onto slides using mounting medium as adhesive
4. staining: dissolve on paraffin with xylol/toluol then rehydrate with ascending alcohol
concentrations; stain with hematoxylin in water and eosin in alcohol (after dehydration
again); cover with cover slip after using nonaqueous mounting medium
formalin does not preserve all cell and tissue components:
H&E adequately display general structure feature, but can’t reveal specific chemical
composition of cell components
Many components/structures are lost during prepping of specimen so need other fixation
methods to show
Alcohols + organic solvents remove neutral lipids (eg. In adipose tissue); must use frozen
sections of formalin-fixed tissue + dyes that dissolve in fat
To retain membrane structures, must use heavy metal fixatives that bind to phospholipids
(permanganate, osmium)
oEg. Osmium tetroxide for EM to preserve membranes in electron micrographs
H&E don’t show elastic material, reticular fibers, basement membranes, lipids  need other
staining procedures
Specific chemical procedures can provide information about the function of cells and the
extracellular components of tissues
oProcedures include: specific binding of a dye, fluorescent dye-labeled antibodies
with a particular cell component, inherent enzymatic activity of a cell component,
autoradiography (radioactively tag precursors of molecules which get incorporated
by cells/tissues before fixation)
oCan be used with light microscopy and EM
Chemical Composition of Histologic Samples

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Notes on Chapter 1
After fixation, the cell components left are usually the large molecules that do not readily
dissolve (usually macromolecules)
Large macromolecular complexes include:
oNucleoproteins: nucleic acids bound to protein
oIntracellular cytoskeletal proteins: complexed w/ associated proteins
oExtracellular proteins: in large insoluble aggregates bount to similar molecules by
cross-linking of neighbouring molecules (eg. Collagen fibers)
oMembrane phospholipid-protein (or carbohydrate) complexes
Large remaining macromolecules make up the formed elements of the tissue and the
displayed structural elements usually are also the functional unit
Small proteins and small nucleic acids are usually lost during preparation of tissue (eg.
tRNA)
Large components lost during fixation with an aqueous fixative include: glycogen and
proteoglycan + glycosaminoglycan
oCan be preserved by using non-aqueous fixative for glycogen; add specific binding
agents to fixative solution to preserve extracellular carbohydrate-containing
molecules
Neutral lipids dissolved by organic solvents during preparation
Large molecules can by lost by hydrolysis from bad pH of fixative
Soluble components, ions, small molecules also lost during preparation of paraffin sections
oGlucose, sodium, chloride, similar substances are lost
oDo not make up formed elements of tissue, just participate in synthetic processes
or cellular reactions
oCan be preserved but will lose structural integrity of proteins; their preservation
provides really great info about the processes they are involved in
Chemical Basis of Staining
Acidic dyes (eg. Eosin) carry net negative charge on coloured part [Na+dye-]
Basic dyes carry net positive charge on coloured part [dye+Cl-]
Hematoxylin has properties that closely resemble basic dyes
oReact with anionic (negative) components of cells/tissues via electrostatic linkages

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Notes on Chapter 1
oPhosphate groups of nucleic acids, sulfate groups of glycosaminoglycans, carboxyl
groups of proteins
oBasophilia: ability of anionic groups to react with basic dye
oReaction of anionic group varies with pH
pH > 10, all 3 anionic groups react w/ basic dyes
5 <pH < 7, sulfate + phosphate groups are ionized and can react with
basic dyes
pH < 4, only sulfate groups remain ionized and can react with basic dyes
oChoosing a specific pH = can visualize specific macromolecules of cell that the
anionic groups make up
Hematoxylin, not really a basic dye, needs mordant (intermediate b/w dye and tissue that
gives hematoxylin properties of basic dyes)
oNot simple electrostatic linkage that will dissociate in water
oNormal basic dissociate after washing with water
oHematoxylin good if you want to stain with acidic dye after
Acidic dyes react with cationic (positive) groups in cells/tissues, particularly with ionized
amino groups of proteins
oAcidophilia: reaction of cationic groups w/ acidic dyes
oNot as specific or precise as basic dyes
oMain factor of primary binding by acidic dyes is electrostatic linkage, but there are
other factors
oCan be used to selectively stain different constituents different colours due to
factors of size, degree of aggregation of dye molecules, permeability and
compactness of tissue
Eg. Mallory staining: 3 different stains of different colours used to stain
collagen, cytoplasm, RBC, nuclei different colours
Eg. Hematoxylin stains nuclei first, then acidic dyes stain cytoplasm/ECM
selectively
Basic dyes can also be used to stain in combination
Basophilic cell + ECM components:
oHeterochromatin + nucleoli of nucleus (ionized phosphate groups of nucleic acids)
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