Laboratory Stains Flashcards
Chromophore
chemical group that results in colour
chromogen
uncoloured molecule + chromophore
auxochrome
molecular structure that attaches dye to tissue
does not have colour itself
two components that make a stain
auxochrome and chromogen
function of mordant
links dye to tissue when auxochrome is weak
Iron of Alum
components of a dye lake
dye + mordant
Regressive Stain
tissue is overstrained
excess stain is removed via differentiation/decolourizing agent
Progressive Stain
tissue is placed in stain for PREDETERMINED amount of time to desired intensity
(no differentiation step)
Hematoxylin & Eosin theory
Regressive stain
Mechanism: chemical ionic bonding
Hematoxylin & Eosin procedure
1) Harris’ Hematoxylin
2) Acid Alcohol
3) Lithium Carbonate
4) Eosin
Periodic Acid Schiff theory
Progressive stain
Mechanism: chemical covalent bonding
Periodic Acid Schiff procedure
Two classes of auxochromes
Anionic (acid, neg charge)
Cationic (basic, pos charge)
Cationic dye
attach to anionic groups in tissue Stain - basic Substrate - basophilic Stain - acidophilic Nuclei, basophil/mast granules, cartilage, RNA
Crystal Violet
Anionic dye
attach to cationic groups stain- acidic substrate - acidophilic stain - basophilic Stain cytoplasm and extracellular structures
Eosin
Mordant Dyes
weak autochrome (need mordant to bridge)
IRON and ALUM
stain indirectly (via bridging)
Dye lakes - highly basic (+) cationic dyes
Solvent Dyes (Lysochromes)
Lacks autochrome
Is hydrophobic
DISSOLVES INTO FATTY AREAS - selective/preferential solubility
Oil Red-O, Sudan Black
Polychromatic Dyes
Spontaneously forms other dyes in solution
Rapid/interoperative diagnosis of cryostat sections
Methylene blue (oxidized into Azure A and Azure B)
Neutral Stain Solutions
Interaction between anionic and cationic dyes - single solution
Romanowsky dyes (heme)
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)
- Natural
- light, air, UV
Long time - 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
