HIS Special Stains Flashcards
(114 cards)
Trichrome stains
- differentiates CONNECTIVE TISSUE from other elements
- uses three ACIDIC dyes*
- based on porosity: smallest dye stains all tissue elements; larger dyes “evict” smaller molecules in larger pores
*NOTE: alum hematoxylin is unsuitable because it is differentiated in acidic pH
Which hematoxylin is used in Trichrome stains ? Why ?
Weigert’s (iron) hematoxylin; alum hematoxylin is differentiated by acidic pH
Principle: Masson Trichrome
- Bouin’s fluid at 50°C for 1 hour
- Biebrich scarlet + Acid fuchsin = STAINS all ACIDOPHILIC elements RED
- Phosphomolybdic/tungstic = colorless dye “evicts” red stain from larger pores (ie. collagen)
- Analine Blue = largest dye stains collagen BLUE
Color of cell components in Masson Trichrome
Collagen: Blue
Nuclei: grey to black
Cytoplasm, keratin, muscle fibre: red
Cause+Troubleshoot: Masson Trichrome stain all decreased
Cause: failure to pre-mordant with Bouin’s when using formalin-fixed tissue
Troubleshoot: pre-mordant for 1 hour at 60°C
What can be used in place of alanine blue for collagen staining in Masson Trichrome ?
Light green, SF yellowish
T or F: In Masson Trichrome, the nuclear stain does not have to be very dark or demonstrate fine chromatin patterns
TRUE; the nuclear stain is for contrast and orientation; it does not have to be very dark or demonstrate fine chromatin patterns
Why should little spills of picric acid from Bouin’s fluid be wiped away immediately ?
Dry picric acid is explosive
2 stains to demonstrate elastin
- Verhoeff’s Van Gieson
- Gomori’s aldehyde fuchsin
Principle: Verhoeff’s Van Gieson
- tissue is OVER-STAINED with IODINE/ FERRIC CHLORIDE/ HEMATOXYLIN
Ferric Chloride = mordant + oxidizer
Iodine = oxidizes hematoxylin and elastin - differentiate using EXCESS FERRIC CHLORIDE = excess mordant breaks tissue-mordant complex*
- iodine is removed by sodium thiosulfate (aka HYPO)
- Van Gieson = counterstain
*NOTE: elastin has the highest affinity for verhoeff and would be decolorized last
Color of cell components in Verhoeff Van Gieson
Elastin: Black
Collagen: Red
Muscle: Yellow
In Verhoeff Van Gieson, the collagen fibres should remain __ during differentiation to avoid removal of stain from elastin fibres
In Verhoeff Van Gieson, the collagen fibres should remain PALE GREY during differentiation to avoid removal of stain from elastin fibres
What must be done after differentiating with Ferric Chloride in Verhoeff Van Gieson staining ?
- Rinse in distilled water to stop differentiation
- Slides must be checked microscopically (collagen must remain pale grey)
3 functions of Ferric Chloride in Verhoeff’s
- Mordant
- Oxidizer
- Differentiator
Verhoeff and Gomori: progressive or regressive ?
Verhoeff = regressive
Gomori’s Aldehyde Fuchsin = progressive
Principle: Gomori’s Aldehyde Fuchsin
- ACIDIFIED PARALDEHYDE + alcoholic BASIC FUCHSIN = RIPEN for 2-4 DAYS before staining
- progressive
- (optional) Potassium permanganate = intensifies staining
- demonstrates elastin
Color of cell components in Gomori’s Aldehyde Fuchsin
Elastin (and beta granules in Pancreas, mast cells, sulfated mucins and cartilage): Purple
Cytoplasm: Green
Storage temp of ripened Gomori’s aldehyde fuchsin
4°C
Although Gomori’s Aldehyde Fuchsin is intended to be used progressively, it can be differentiated with __ if desired.
Although Gomori’s Aldehyde Fuchsin is intended to be used progressively, it can be differentiated with 70% ALCOHOL if desired.
What kind of stains demonstrate Reticulin ?
Silver stains
Principle: Gordon and Sweet’s Reticulin Stain
- potassium permanganate oxidizes Reticulin
- oxalic acid bleaches discolouration from permanganate
INDUCED ARGYROPHILIA:
- Ferric ammonium sulfate = sensitizer binds to reticulin; to be replaced by silver solution
- formalin reduces silver to metallic form
- gold chloride tones silver = brown to black
- sodium thiosulfate removes any unreduced silver*
- counterstain: nuclear fast red or eosin
*NOTE: prevents section from darkening when exposed to light
Color of cell components in Gordon&Sweet’s Reticulin Stain
Reticulin: Black
Cytoplasm: Red
During Gordon&Sweet’s Reticulin staining, a white precipitate forms on the walls of the jar/ solution surface. What does this mean ? How will this affect staining ?
- glassware was NOT acid washed/ bleached + rinsed with dH2O
- silver solution is CONTAMINATED
- staining will be weak
Why are Reticulin silver stains usually made fresh ?
Ferric AMMONIUM sulfate + SILVER = EXPLOSIVE
- discard after use
- avoid sunlight