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33 Cards in this Set

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the most commonly studied specimen preparation
formalin-fixed, paraffin-embedded, and hematoxylin & eosin (H&E)-stained
4 things fixation does
-terminate cell metabolism
-prevent enzymatic degradation of cells and tissues
-kill pathogenic microorganisms
-harden tissue by cross-linking or denaturing protein molecules
1 picometer (pm) = ? angstrom (A)
0.01 angstrom (A)
1 angstrom = ? nanometer (nm)
0.1 nm
10 angstroms = ? nanometer (nm)
1.0 nm
1 nanometer = ? picometers
1,000 picometers
1,000 nanometers = ? micrometers (um)
1.0 um
1,000 micrometers = ? millimeters (mm)
1.0 mm
caveats to H&E stained sections of formalin specimens
-cannot highlight chemical composition of cell components
-many components are lost in the prep process
-elastic material, reticular fibers, basement membranes, and lipids (all of which are structural components ofhistological sections)
solvents used to remove neutral lipids in routine preparations
alcohols and organic solvents
solvents used to retain neutral lipids in routine preparations
frozen sections of formalin-fixed tissues and dyes that dissolve in fats
solvents used to retain membrane structures
special fixatives containing heavy metals that bind to the phosphoplipids (such as permanganate and osmium tetroxide)
purpose of using H&E staining
displays most structural features
stain used to reveal the structural component: elastic material
orcein and resorcin-fuchsin
stain used to reveal the structural components: reticular fibers and basement membrane material
silver impregnation
3 steps in preparation of a tissue or organ sample
1. Fixation
2. Embedding
3. Staining
preparation of a tissue or organ sample to preserve structure
fixation
Most commonly used fixative
Formalin, a 37% aqueous sltn of formaldehyde buffered with phosphates
13 steps in the permanent preparation of tissue
1. fixation with formalin
2. wash
3. dehydrated with ascending [OH] up to 100% OH (to remove H2O)
4. wash with organic slvnts (xylol or toloul) (to remove OH)
5. infiltration of sample with melted paraffin
6. sliced in microtome (5-15um)
7. sections mounted on slides (albumin adhesive)
8. dissolve paraffin with orgo slvnts (xylol or toluol)
9. rehydrate with descending [OH]
10. stain with hematoxylin in H2O(blue--nuclear DNA/cytoplasmic RNA)
11. dehydrate with ascending [OH]
12. stain w eosin in OH (red--counterstain)
13. wash with orgo slvnts (xylol or toluol) to mounting and cover
incorporation of radioactively tagged precursors of cell molecule before fixation
autoradiography
3 steps in frozen section preparation (~10 min)
1. freeze tissue sample w compressed CO2 or immersed in isopentane (-50C) or in high-efficiency fridge
2. microtome sectioned within a cryostat (5-10 um) and mounted
3. Stain to separate nuclei from other cell parts using H&E, methylene blue, or PAS stains
macromolecular complexes that remain after fixation
-nucleoproteins (nucleic acids bound to protein)
-intracellular cytoskeletal proteins
-extracellular proteins in big, insol, cross-linked complexes
-membrane phospholipid/carbohydrate complexes
tissue components usually retained in preparation
-nucleic acids
-proteins
-phospholipids
tissue components usually lost in preparation
-small proteins
-small nucleic acids (tRNA)
-neutral lipids
-glycogen (intracell. storage carb in liver & muscle cells)
-proteoglycans & glycosaminoglycans (extracell. carb. in conn. tiss.)
ways to preserve molecules lost due to use of aqueous fixative
-use of nonaqeous fixative for glycogen
-adding specific binding agents to fixative sltn to preserve extracell. carb. components in molecules
reason to preserve soluble components, ions, and small molecules (interm. metabolites, glucose, sodium, chloride) lost in prepartion
to get info about cell metabolism, active transport, other cellular processes
[ Na+ Dye- ]
Acidic Dye:
-carries a net negative charge on the dye portion
-react with cationic (positive) groups in cells and tissues (particularly w the ionized amino groups of proteins)
[ Dye+ Cl- ]
Basic Dye:
-carries net positive charge on the dye portion
-react with the anionic (negative) components of cells and tissues (such as phosphate grps of nucleic acids, sulfate grps of glycosaminoglycans, & carboxyl grps of proteins)
Basic Dyes
Methyl green (green)
Methylene blue (blue)
Pyronin G (red)
Toulidine blue (blue)
Acidic Dyes
Acid fuchsin (red)
Aniline blue (blue)
Eosin (red)
Orange G (orange)
ability of anionic grps to react w basic dye
basophilia
ability of cationic grps to react w acidic dye
acidophilia
an intermediate link between the tissue component and the dye
mordant