Special Stains knowledge Flashcards
H&E for
Progressive vs Regressive
Most commonly used routine stains
Needs mordant (alum, iron, tungsten) since tissue is anionic and haematin is anionic
Haematoxylin is responsible for nuclear staining - Blue
Eosin is responsible for Cytoplasmic staining - Shades of pink
Progressive staining is a slower staining process in which the tissue is left in the staining solution just long enough to reach the desired endpoint.
Regressive staining is a more rapid staining process in which the tissue is deliberately overstained and then de-stained with a weak acid alcohol until the desired point of staining is reached.
Alcian blue for
Principle
Components
Results
Used to distinguish between neutral and acid mucins. By Altering the pH of the Alcian solution the subtypes of acidic mucins can be characterised. Used to identify Barrett’s epithelium and for intestinal metaplasia in gastric biopsies.
Components
Alcian Blue
Iron A and B Hx = Equal parts = Weigerts Iron hx
VG solution
Results
Acid Mucin - Blue
Nuclei - Dark grey
Collagen - Red / Pink
Other Structures - Yellow
Hales Colloidal Iron (Mowry Method) for
Principle
Components
Results
Principle
At Low pH, colloidal iron will be absorbed into anionic tissue. The absorbed iron is visualized via Perls Prussian blue method (conversion to ferric ferrocyanide) Can be combined with PAS for a more sensitive and intense method than ABPAS.
Components
Colloidal Iron stock solution (Distilled water and Ferric chloride)
Colloidal Iron working solution (add distilled water and acetic acid)
Potassium ferrocyanide / HCl solution
Nuclear Fast Red - Counterstain
Results
Acid mucins - Blue
Background - Red
Wade Fide for
for Mycobacterium Leprae
The same principle and result as ZN
only different component is the use of sulphuric acid as a differentiator.
Orcein for
Principle
Components
Results
for HepB surface antigen and Elastic fibers
Principle
Tissue sections oxidised with acidic potassium permanganate causing HepB SA to become reactive residues. These are free to react with orcein stain to give a ‘ground glass’ appearance to affected hepatocytes. (not a specific stain for HepB)
Components
Acidified potassium permanganate
Orcein solution (Synthetic orcein, 70 alcohol and conc HCl)
1% Oxalic acid (decolouriser)
Results
HepB Antigen - Burgundy/brown
Elastic - Burgundy/brown
Background - Light brown
Gram (Twort) for
Principle
Components
Results
for Bacteria
Principle
Gram-positive - more cell wall layers = able to trap crystal violet/iodine
Gram-positive - have magnesium ribonuclease in cell wall - forms insoluble complex with crystal violet
Components
Crystal violet
Grams iodine
Tworts counter stain (Neutral red, Fast green, distilled water)
Acetone (decolouriser)
Result
Gram-positive - Dark blue
Gram-negative - Red
Nuclei - Red
Cytoplasm / RBC - Pale Green
Masson Trichrome for
Principle
Components
Result
Muscle, Fibrin, RBCs and Connective tissue
Heated dichromate solution increases the intensity of the stain by making more tissue groups available to react with trichrome dyes.
Smaller dye molecules (Ponceau Fuchsin) penetrate tissue, the PMA (phosphomolybdic acid) then competes with PF and gains access to collagen easily, expelling the dye. Collagen is then free to stain with the Light Green.
Components
Acid dichromate solution ( sulphuric acid, potassium dichromate)
Ponceau Fuchsin solution (Acid fuchsin in acetic acid)
Phosphomolybdic acid - Differentiator
Light green in acetic acid - counter stain
Result
Muscle, RBC, Fibrin - Red
Connective tissue - Green
Nuclei - Dark blue
MSB for
Principle
Components
Result
Martius, Scarlet and Blue for Fibrin
Principle
This technique employs a small molecule of yellow dye which when used with phosphotungstic acid in an alcoholic solution will selectively stain red cells.
Phosphotungstic acid blocks the staining of other tissue structures however it is possible that early fibrin may take up the dye.
A medium-sized molecule of red dye is then used to stain muscle and mature fibrin. The staining of collagen is still prevented by phosphotungstic acid.
A large molecule of blue dye is then applied, this will stain collagen and old fibrin.
Components
Martius Yellow - Small dye
Brilliant crystal scarlet - Medium dye
Aniline Blue solution - Large dye
Iron alum
Phosphotungstic acid
Acetic acid
Results
Nuclei - Blue/ Black
RBC, Early fibrin - Yellow
Muscle- Red
Collagen and Old Fibrin - Blue
Fibrin - Red
EVG for
Result
Van Gieson for Elastic fibres
Elastic Fibres - Dark Blue
Collagen - Red
RBC’s - Yellow
Gordon and Sweets for
Principle
Components
Result
Reticulin Fibres - Argentaffin
Principle
Impregnation of retic fibres with silver deposits occurs via oxidation and reduction reactions.
Reticulin has low affinity for silver salts therefore must be oxidized with potassium permanganate and sensitised using iron alum which targets and binds to the retic.
An ammonical silver solution removes and replaces iron alum and impregnates retic fibres.
10% formalin is then applied to highlight silver deposits on the retic. Gold chloride is used to improve deposit stability fibre contrast and clarity.
Unreduced silver and excess gold are removed using sodium thiosulphate
Components
Ammonical Silver solution ( 10% silver nitrate, ammonium hydroxide, 10% potassium hydroxide)
Acidified permanganate
Oxalic acid
Iron Alum
10% Formalin
Gold chloride
Sodium thiosulphate
Nuclear Fast red - counterstain
Result
Reticulin fibres - Black
Nuclei - Red
Collagen and background - Pink / Grey
What is reticulin?
Fine delicate fibre is connected to coarser fibres that provide the supporting framework in highly cellular organs like the spleen, liver and lymph nodes.
Von Kossa for
Principle
Components
Results
Calcium
Principle
Silver is substituted for calcium in the form of calcium salts which are then reduced to black metallic silver by the use of UV light.
Components
Silver nitrate
Sodium thiosulphate
Neutral Red - Counterstain
Results
Calcium deposits - Black
Background - Pink
Nuclei - Red
Rhodanine for
Principle
Components
Results
Copper
Principle
Rhodanine forms chelate bonds with copper
Components
Rhodanine stock solution
Rhodanine working solution (add distilled water)
0.5% borax solution
Counterstain - Harris Hx
Results
Copper - Red
Nuclei - Blue
Bile - Green
Fontana Masson for
Principle
Components
Results
Melanin
Principle
Melanin has argentaffin properties which reduces ammonical silver nitrate to metallic silver without the use of an external reducing agent.
Components
Ammonical silver solution
Gold chloride - Tone
Sodium thiosulphate - Fix
Neutral Red - Counterstain
Results
Melanin - Black
Nuclei - Red
Oil Red O for
Lipids
Principle
Fresh tissues are necessary because fixatives containing alcohol or routine tissue processing with clearing will remove lipids.
Components
Working Oil red O solution
Harris Hx - counterstain
Aqueous mountant
STWS
NB. No dehydration or clearing
Results
Lipids - Red - Orange
Nuclei - Blue
