Histology

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the most commonly studied specimen preparation

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formalin-fixed, paraffin-embedded, and hematoxylin & eosin (H&E)-stained

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4 things fixation does

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-terminate cell metabolism
-prevent enzymatic degradation of cells and tissues
-kill pathogenic microorganisms
-harden tissue by cross-linking or denaturing protein molecules

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1 picometer (pm) = ? angstrom (A)

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0.01 angstrom (A)

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1 angstrom = ? nanometer (nm)

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0.1 nm

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10 angstroms = ? nanometer (nm)

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1.0 nm

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1 nanometer = ? picometers

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1,000 picometers

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1,000 nanometers = ? micrometers (um)

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1.0 um

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1,000 micrometers = ? millimeters (mm)

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1.0 mm

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caveats to H&E stained sections of formalin specimens

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-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)

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solvents used to remove neutral lipids in routine preparations

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alcohols and organic solvents

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solvents used to retain neutral lipids in routine preparations

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frozen sections of formalin-fixed tissues and dyes that dissolve in fats

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solvents used to retain membrane structures

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special fixatives containing heavy metals that bind to the phosphoplipids (such as permanganate and osmium tetroxide)

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purpose of using H&E staining

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displays most structural features

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stain used to reveal the structural component: elastic material

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orcein and resorcin-fuchsin

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stain used to reveal the structural components: reticular fibers and basement membrane material

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silver impregnation

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3 steps in preparation of a tissue or organ sample

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1. Fixation
2. Embedding
3. Staining

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preparation of a tissue or organ sample to preserve structure

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fixation

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Most commonly used fixative

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Formalin, a 37% aqueous sltn of formaldehyde buffered with phosphates

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13 steps in the permanent preparation of tissue

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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

Front 20

incorporation of radioactively tagged precursors of cell molecule before fixation

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autoradiography

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3 steps in frozen section preparation (~10 min)

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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

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macromolecular complexes that remain after fixation

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-nucleoproteins (nucleic acids bound to protein)
-intracellular cytoskeletal proteins
-extracellular proteins in big, insol, cross-linked complexes
-membrane phospholipid/carbohydrate complexes

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tissue components usually retained in preparation

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-nucleic acids
-proteins
-phospholipids

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tissue components usually lost in preparation

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-small proteins
-small nucleic acids (tRNA)
-neutral lipids
-glycogen (intracell. storage carb in liver & muscle cells)
-proteoglycans & glycosaminoglycans (extracell. carb. in conn. tiss.)

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ways to preserve molecules lost due to use of aqueous fixative

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-use of nonaqeous fixative for glycogen
-adding specific binding agents to fixative sltn to preserve extracell. carb. components in molecules

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reason to preserve soluble components, ions, and small molecules (interm. metabolites, glucose, sodium, chloride) lost in prepartion

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to get info about cell metabolism, active transport, other cellular processes

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[ Na+ Dye- ]

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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)

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[ Dye+ Cl- ]

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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)

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Basic Dyes

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Methyl green (green)
Methylene blue (blue)
Pyronin G (red)
Toulidine blue (blue)

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Acidic Dyes

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Acid fuchsin (red)
Aniline blue (blue)
Eosin (red)
Orange G (orange)

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ability of anionic grps to react w basic dye

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basophilia

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ability of cationic grps to react w acidic dye

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acidophilia

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an intermediate link between the tissue component and the dye

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mordant