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Histology (MLSCI 250) > Laboratory Stains > Flashcards

Laboratory Stains Flashcards

1
Q

Chromophore

A

chemical group that results in colour

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2
Q

chromogen

A

uncoloured molecule + chromophore

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3
Q

auxochrome

A

molecular structure that attaches dye to tissue

does not have colour itself

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4
Q

two components that make a stain

A

auxochrome and chromogen

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5
Q

function of mordant

A

links dye to tissue when auxochrome is weak

Iron of Alum

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6
Q

components of a dye lake

A

dye + mordant

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7
Q

Regressive Stain

A

tissue is overstrained

excess stain is removed via differentiation/decolourizing agent

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8
Q

Progressive Stain

A

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

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9
Q

Hematoxylin & Eosin theory

A

Regressive stain

Mechanism: chemical ionic bonding

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10
Q

Hematoxylin & Eosin procedure

A

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

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11
Q

Periodic Acid Schiff theory

A

Progressive stain

Mechanism: chemical covalent bonding

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12
Q

Periodic Acid Schiff procedure

A
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13
Q

Two classes of auxochromes

A

Anionic (acid, neg charge)

Cationic (basic, pos charge)

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14
Q

Cationic dye

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

Crystal Violet

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15
Q

Anionic dye

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

Eosin

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16
Q

Mordant Dyes

A

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

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17
Q

Solvent Dyes (Lysochromes)

A

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

Oil Red-O, Sudan Black

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18
Q

Polychromatic Dyes

A

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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19
Q

Neutral Stain Solutions

A

Interaction between anionic and cationic dyes - single solution

Romanowsky dyes (heme)

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20
Q

Leuko Dyes

A

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

21
Q

Metachromatic dye

A

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

22
Q

Physical Factors

A

Absorption

  • dimension of surface area
  • density
  • permeability
  • size of dye molecule
23
Q

Chemical Factors

A 
Electrocovalent bonds
covalent bonds
hydrogen bonds
van der waals forces
hydrophobic interaction
24
Q

Covalent Bonds

A

2 atoms sharing electrons
STRONG, hard to correct
organi chemicals

Schiff’s reagent, Verhoeff’s stain

25
Q

Hydrogen Bonds

A

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

26
Q

Van der Waals

A

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

Aldehyde fuchsin

27
Q

Hydrophobic Interaction

A

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

28
Q

Metallic impregnation

A

Silver in an alkaline solution readily precipitates

3 types: argentaffin, argyrophil, metallic substitution

29
Q

Argentaffin

A

natural occurrence

reactive groups reduce silver salts

30
Q

Argyrophil

A

force it to occur

tissue elements not reactive enough to reduce silver, requires outside reducer

31
Q

metallic substitution

A

ion exchange

32
Q

Progressive Stain

A

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

Periodic Acid Schiff

33
Q

Regressive

A

Tissue is overstained

excess stain is removed using differentiation or decolourizing step

34
Q

Methods of differentiation

A

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

35
Q

Before staining:

A
  • deparaffinization (barrier)
  • bring slides to water (opposite of processing)
  • — xylene removes wax, alcohol removes xylene
36
Q

Oxidation (ripening)

A
  1. Natural
    - light, air, UV
    Long time
  2. Chemical
    - quick
    - easy to overdo
37
Q

Immunohistochemistry

A

Detection of Ag on cells using Ag-Ab rxn

38
Q

Rationale for IHC

A
  • pathological Ag
  • lineage to cells/tumors (metastasis vs new)
  • stage/grade of cancers
  • Personalized.precise medical treatment
  • distribution/localization of Ag in tissue
39
Q

Polyclonal Ab

A

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

40
Q

Monoclonal Ab

A

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

41
Q

Primary Ab

A

Binds DIRECTLY to Ag

usually less of a signal

42
Q

Secondary Ab

A

Binds to primary Ab
INDIRECT fromAg
Signal is typically amplified

43
Q

Labelling and Detection (IHC)

A 
Enzyme (HRP) or polymer bound to Ab (3rd layer)
Chromogen added 
--> DAB (brown)
--> AP (red)
Counterstain (hematoxylin)
44
Q

Third layer (IHC)

A 
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)
45
Q

Immunoflourescence

A

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

46
Q

Problems with immunoflourescence

A

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

47
Q

methods of Epitope retrieval

A

** Heat-Induced Epitope Retrieval (HIER)

Enzyme0Induced Epitope Retrieval (EIER)

48
Q

Electrocovalent Bonds (ionic, electrostatic, salt linkage)

A

Between oppositely charged ions
most common

Schiff’s reagent
Verhoeff’s Stain

Histology (MLSCI 250) (18 decks)

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