Laboratory Stains Flashcards by Mikayla Steinbach

Chromophore

chemical group that results in colour

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chromogen

uncoloured molecule + chromophore

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auxochrome

molecular structure that attaches dye to tissue

does not have colour itself

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two components that make a stain

auxochrome and chromogen

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function of mordant

links dye to tissue when auxochrome is weak

Iron of Alum

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components of a dye lake

dye + mordant

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Regressive Stain

tissue is overstrained

excess stain is removed via differentiation/decolourizing agent

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Progressive Stain

tissue is placed in stain for PREDETERMINED amount of time to desired intensity
(no differentiation step)

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Hematoxylin & Eosin theory

Regressive stain

Mechanism: chemical ionic bonding

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Hematoxylin & Eosin procedure

1) Harris’ Hematoxylin
2) Acid Alcohol
3) Lithium Carbonate
4) Eosin

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Periodic Acid Schiff theory

Progressive stain

Mechanism: chemical covalent bonding

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Periodic Acid Schiff procedure

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Two classes of auxochromes

Anionic (acid, neg charge)

Cationic (basic, pos charge)

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Cationic dye

attach to anionic groups in tissue
Stain - basic
Substrate - basophilic
Stain - acidophilic
Nuclei, basophil/mast granules, cartilage, RNA

Crystal Violet

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Anionic dye

attach to cationic groups
stain- acidic
substrate - acidophilic
stain - basophilic
Stain cytoplasm and extracellular structures

Eosin

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Mordant Dyes

weak autochrome (need mordant to bridge)
IRON and ALUM
stain indirectly (via bridging)
Dye lakes - highly basic (+) cationic dyes

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Solvent Dyes (Lysochromes)

Lacks autochrome
Is hydrophobic
DISSOLVES INTO FATTY AREAS - selective/preferential solubility

Oil Red-O, Sudan Black

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Polychromatic Dyes

Spontaneously forms other dyes in solution
Rapid/interoperative diagnosis of cryostat sections

Methylene blue (oxidized into Azure A and Azure B)

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Neutral Stain Solutions

Interaction between anionic and cationic dyes - single solution

Romanowsky dyes (heme)

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Leuko Dyes

Leuko - colourless
Chromophores are easily REDUCED
chemical run occurs with reactive tissue groups
reversible

Metachromatic dye

dye tissues different color than the dye
tissue components - chromotropes
Aqueous mounting media
Sulfation - induced metachromasia

Physical Factors

Absorption

dimension of surface area
density
permeability
size of dye molecule

Chemical Factors

Electrocovalent bonds
covalent bonds
hydrogen bonds
van der waals forces
hydrophobic interaction

Covalent Bonds

2 atoms sharing electrons
STRONG, hard to correct
organi chemicals

Schiff’s reagent, Verhoeff’s stain

Hydrogen Bonds

attraction b/w: lone pair electrons on O or N 7 nucleus of H Weakers than covalent or ionic affects staining in non aqueous solvents

Van der Waals

electrostatic attraction between electrons of one atom and nucleus of another Short range forces Weak Aldehyde fuchsin

Hydrophobic Interaction

non-polar molecule affinity for another Aqueous mounting media dye must be more soluble in substrate and solvent

Metallic impregnation

Silver in an alkaline solution readily precipitates | 3 types: argentaffin, argyrophil, metallic substitution

Argentaffin

natural occurrence | reactive groups reduce silver salts

Argyrophil

force it to occur | tissue elements not reactive enough to reduce silver, requires outside reducer

metallic substitution

ion exchange

Progressive Stain

use each stain until desired effect is achieved usually pre-determined amount of time no differentiation step Periodic Acid Schiff

Regressive

Tissue is overstained | excess stain is removed using differentiation or decolourizing step

Methods of differentiation

Washing (water, alcohol, solvent) Excess mordent Oxidizing agent other dyes

Before staining:

- deparaffinization (barrier) - bring slides to water (opposite of processing) - --- xylene removes wax, alcohol removes xylene

Oxidation (ripening)

1. Natural - light, air, UV Long time 2. Chemical - quick - easy to overdo

Immunohistochemistry

Detection of Ag on cells using Ag-Ab rxn

Rationale for IHC

- pathological Ag - lineage to cells/tumors (metastasis vs new) - stage/grade of cancers - Personalized.precise medical treatment - distribution/localization of Ag in tissue

Polyclonal Ab

Ag injected into animal Ag activates B cells Plasma B cells produce POLYCLONAL Ab (mixed population/variety) Obtain antiserum from rabbit that has polyclonal Ab Less $ Binds to multiple areas of target Ag

Monoclonal Ab

Animal injected with Ag Collect specific spleen cells myeloma cells fuse with specific spleen cell HYBRIDOMA - replication of a single specific Ab Very $$$ Binds to specific Ag only

Primary Ab

Binds DIRECTLY to Ag | usually less of a signal

Secondary Ab

Binds to primary Ab INDIRECT fromAg Signal is typically amplified

Labelling and Detection (IHC)

``` Enzyme (HRP) or polymer bound to Ab (3rd layer) Chromogen added --> DAB (brown) --> AP (red) Counterstain (hematoxylin) ```

Third layer (IHC)

``` Allows for smaller amounts of 1° and 2° Increases sensitivity - PAP (peroxidase anti-peroxidase) - ABC (Avidin-biotin complex) uses biotinylated 2°Ab - LSAB (labeled streptavidin-biotin) ```

Immunoflourescence

FROZEN tissue sections Flourophore-linked 2°/1° Ab Can use multiple labels

Problems with immunoflourescence

Fixation cannot be delayed (destroy proteins/Ag) Formalin (routine fixative) cross-links Ag epitope Skilled microtomy - need to collect next tissue section

methods of Epitope retrieval

** Heat-Induced Epitope Retrieval (HIER) | Enzyme0Induced Epitope Retrieval (EIER)

Electrocovalent Bonds (ionic, electrostatic, salt linkage)

Between oppositely charged ions most common Schiff's reagent Verhoeff's Stain