Modified Movat's Pentachrome Stain
Fixation: 10% buffered neutral formalin
Sections: 6u paraffin sections
Solutions:
1% Alcian Blue Solution ( Stable for 2 months )
Alcian blue, 8 GS 1.0 g
DdH2O 100 ml
Glacial acetic acid 1 ml
Alkaline Alcohol (pH 8 or above )
Ammonium hydroxide 10 ml
Alcohol 95% 90 ml
Hematoxylin Solution (Verheoff's) (Stable for several months)
Absolute alcoholic hematoxylin, 10% 25 ml
Absolute alcohol 25 ml
Ferric chloride, 10% aqueous (Make fresh) 25 ml
Iodine solution 25 ml
Iodine 2 g
Potassium iodide 4 g
DdH2O 100 ml ( Dissolve 4g Potassium iodide
in 4-8ml ddH2O, then add 2g Iodine. After Iodine is dissolved dilute to 100ml).
Sodium Thiosulfate
Sodium Thiosulfate 5 g
DH2O 100 ml
Crocein Scarlet-AcidFuchsin Solution
Stock A
Crocein Scarlet moo 7B 0.1 g
ddH2O 99.5 ml
Glacial acetic acid 0.5 ml
Stock B
Acid fuchsin 0.1 g
ddH2O 99.5 ml
Glacial acetic acid 0.5 ml
Working Solution:
Stock A 8 parts
Stock B 2 parts
0.5% acetic acid
20% acetic acid 25 ml
ddH2O 975 ml
5% aqueous phosphotungstic acid
Phosphotungstic acid 10g
ddH2O 200ml
(To remove the crocein scarlet-acid fusin stain from the extra cellular connective tissue and ground substance. When the solution is removed the staining with insoluble monastral fast blue is again shown.)
Alcoholic Saffron Solution
Saffron (Safran du Gatinais 6 g
Absolute alcohol 100 ml
(Keep tightly closed to prevent hydration. Incubate at 56-58°C for 48 hours.)
Procedure
- Deparaffinize and hydrate to ddH2O.
- Stain in alcian blue for 20 min. (Stain for ground substance, mucin-blue).
- Wash in running water for 10 min.
- Place slides in alkaline alcohol for 1-2 hour. (convert the alcian blue into insoluble monastral fast blue.)
- Wash in running water for 10 min. (Complete removal of alkaline alcohol with running water is important. Failure to remove all alkaline alcohol will inhibit the stain that follow.)
- Rinse in dH2O.
- Stain in Verheoff's hematoxylin solution for 15 min. (Stain for nuclei & elastic fiber-black).
Absolute alcoholic hematoxylin, 10% 25 ml
Absolute alcohol 25 ml
Ferric chloride, 10% aqueous (Make fresh) 25 ml
Iodine solution 25 ml - Rinse several changes of dH2O.
- Differentiate in 2% aqueous ferric chloride and agitate slides gently.
(Stop differentiation with several changes of tap water and check microscopically for black elastic fiber staining and gray background. Repeat 2% ferric chloride treatment and tap water rinses as necessary. If elastic fiber staining is too pale, restain in the saved verhoeff's solution.) - Place slides in sodium thiosulfate for one min.(Discard solution)
- Wash in running tap water for 5 min; rinse in dH2O.
- Stain in crocein scarlet-acid fuchsin (8:2)for 1 1/2 min. (Stain for fibrinoid, fibrin-intense red, muscle-red).
- Rinse in several changes of dH2O
- Rinse in 0.5% acetic acid water.
- Differentiate slides in 5% aqueous phosphotungstic acid, 5-10min. (Check microscopically. Continue differentiation until collagen is pale pink in color and the ground substance, initially colored red, becomes bluish in color.)
- Rinse in 0.5% acetic acid water. (To remove phosphotungstic acid).
- Rinse in 100% alcohol X3.
- Stain in Saffron for 15 -20 min. Use a screw capped coplin jar for this step, and keep the jar sealed during the staining procedure. If collagen is not sufficiently yellow, stain for a longer period.( Stain for collagen & reticular fiber-yellow).
- Rinse in 100% alcohol X3 and xylene X2 and mount in mounting mediums.
Results:
Nuclei and elastic fibers: Black.
Collagen and reticular fibers: Yellow.
Ground substance, mucin: Blue.
Fibrinoid, fibrin: Intense red
Muscle: Red
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