Special Stains Flashcards
Define special stains
Staining methods that use dyes, metal impregnation, or chemical reactions to demonstrate tissue structures.
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
Masson Trichrome
- differentiates between SMOOTH MUSCLE vs COLLAGEN in tumors
- demonstrates increased collagen in cirrhosis
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)
- Alanine Blue = largest dye stains collagen BLUE
Color: Nuclei in Masson Trichrome
grey to black
Color: Cytoplasm, keratin, muscle fibre, erythrocytes in Masson Trichrome
Red
Color: Collagen in Masson Trichrome
Blue
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 analine 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
- Gomori’s aldehyde fuchsin
Elastin fibres contain __ bridges, which oxidizes to form __ derivatives. These substances are strongly (acidophilic/ basophilic).
Elastin fibres contain DISULFIDE bridges, which oxidizes to form SULFONIC ACID derivatives. These substances are strongly BASOPHILIC.
Principle: Verhoeff 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: Elastin in Verhoeff Van Gieson
Black
Color: Collagen in Verhoeff Van Gieson
Red
Color: Muscle in Verhoeff Van Gieson
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)
Gomori’s Aldehyde Fuchsin is described as “empirical.” What does this mean ?
- we are unsure of why it stains elastin
- elastin stains with both eosin (anionic) and basic fuchsin (cationic), so it can’t be simply ionic binding
- hydrogen bonding is hypothesized to be a contributor
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
Color: Elastin (and beta granules in Pancreas, mast cells, sulfated mucins and cartilage) in Gomori’s Aldehyde Fuchsin
Purple
Color: Potassium permanganate counterstain in Gomori’s Aldehyde Fuchsin
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 = aldehyde groups of reticulin
- oxalic acid bleaching removes discolouration from permanganate
- (INDUCED ARGYROPHILIA) Ferric ammonium sulfate = sensitizer binds to aldehydes; to be replaced by silver from ammoniacal 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: Reticulin in Gordon&Sweet’s Reticulin Stain
Black
Color: Counterstain in Gordon&Sweet’s Reticulin Stain
Red
During Gordon&Sweet’s Reticulin staining, a white precipitate forms on the walls of the jar/ solution surface. What does this mean ?
- 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
How are silver solutions neutralized ?
- add CHLORIDE ions (HCl, bleach, or saline)
- precipitate out the silver = silver chloride
- dispose in HALOGENATED waste
2 stains to demonstrate carbohydrates
- PAS (Periodic Acid Schiff)
- Alcian blue
Which carbohydrates are PAS positive ?
- glycogens
- NEUTRAL mucins (mucopolysaccharides)
NOTE: basement membrane, fungi (C. albicans), zymogen granules, colloid, and reticulin are also POS
Which carbohydrates are Alcian blue positive ?
(ACID mucopolysaccharides):
- carboxylated mucins
- sulfated mucins
Glycogen: PAS vs PAS-D
PAS = POS
PAS-D = neg
Principle: Periodic Acid Schiff
- periodic acid oxidizes hydroxyl in carbs = aldehydes form
- schiff reagent (basic fuchsin + sulfurous acid*) reacts with aldehydes = bind to tissue
- water rinses removes loose sulfurous acid groups = restores qunoid ring = BRIGHT RED
*NOTE: reduction causes quinoid ring to be lost = colorless
Why is periodic acid used to form aldehydes in PAS ?
Periodic acid is a WEAK OXIDIZER
- others will oxidize hydroxyl beyond aldehydes
What is Schiff reagent made of ?
Basic fuchsin + sulfurous acid
Color: PAS-positive tissue elements
Magenta
Cause+Troubleshoot: PAS Non-specific staining
Cause: did not rinse excess Schiff reagent
Troubleshoot: water rinse
In PAS, __ is important for proper color development
In PAS, the WATER RINSE is important for proper color development
PAS-D
Periodic Acid Schiff with Diastase (α-amylase):
- digestion step = specific for glycogen
Principle: PAS-D
- diastase (α-amylase) breaks down glycogen into SMALLER sugars (maltose + glucose)
- water rinse WASHES AWAY SIMPLE SUGARS
- stained in duplicate: PAS-D vs PAS
- INDIRECT DETECTION = when compared to PAS, absent staining on the digested slide (PAS-D) will be due to glycogen
Color: glycogen on PAS-D
UNSTAINED bc of digestion+rinse
Color: PAS-pos tissue elements on PAS-D
Magenta
NOTE: EXCEPT glycogen
Source of α-amylase for PAS-D
Saliva
Alcian blue
- a copper pthalocynanin CATIONIC dye
- stains ACID MUCINS to differentiate adenocarcinomas
Principle: Alcian blue
- at pH 2.5, IONICALLY binds with carboxylated and sulfated (acid) mucins
- at pH 1.0, only sulfated acid mucins will stain
Color: at pH 2.5, carboxylated/ sulfated mucins in Alcian blue
Dark blue
Color: at pH 1.0, sulfated mucins in Alcian blue
Light blue
Color: Nuclei in Alcian blue
Red
T or F: Alcian blue will appear more lightly stained in a lower pH
TRUE; Alcian blue will appear more lightly stained in a lower pH
- sulfated mucins will be light blue in pH 1.0, but darker at pH 2.5
How is a low, stable pH achieved for Alcian blue staining ?
Acid rinse prior to staining
Alcian Blue + PAS
- Stain Alcian blue first = stains acid mucins BLUE
- PAS = stains neutral mucins MAGENTA
- tissue elements that contain both acid and neutral mucins = purple