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Movat’s Modified Pentachrome Protocol

This is a quick version of Movat’s Pentachrome.  The long version is an overnight stain, I hate waiting till the next day to find out if the stain worked.  It is sometimes hard to visualize all 5 colors but with a aorta in the heart, it is possible.  The stain picture will have to be edited in later.

Control tissue that I use: Skin, Aorta in the heart

Safety equipment: Work under a hood with lab coat, gloves, and glasses.

  1. Deparaffinize and hydrate to DH2O
  2. Preheat Alkaline alcohol in 60C oven or water bath
  3. Mordant in bouin’s at 60C water bath——- 30 minutes (under hood!) (re-use)
  4. Cool—————————————– 10 minutes in room temp water
  5. Wash in running water—————– 10 minutes
  6. 1% Alcian blue————————— 25 minutes (re-use)
  7. Wash in running water—————– 5 minutes
  8. Alkaline alcohol at 60C —————- 10 minutes
  9. Wash in running water—————— 5 minutes
  10. Weigerts———————————– 15 minutes (dump)
  11. Differentiate in 2% ferric chloride for- 3 minutes (re-use)
  12. Rinse in H2O——————————- 10 seconds
  13. Sodium thiosulfate for——————– 1 minute (re-use)
  14. Wash in running water——————- 5 minutes
  15. Crocein scarlet/ Acid fuchsin———- 1.5 minutes (re-use)
  16. Wash in running water—————— 5 minutes
  17. 5% Phosphotungstic acid————– 5 minutes (re-use)
  18. Transfer directly to 1% acetic acid–  5 minutes (re-use)
  19. Wash in running water—————— 5 minutes
  20. Dehydrate quickly 95% alc. 1 min/2x    100% alc. 1 min
  21. Alcoholic saffron————————– 60 minutes (re-use)
  22. Rinse in 2 changes of 100% alcohol, 1 min each
  23. Clear in xylene and mount

Results:

Nuclei and elastic fibers—Black

Collagen—Yellow

Ground substance and mucin–Blue

Fibrinoid, fibrin–Intense Red

Muscle–Red

 

An Expert Histology Service

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Masson’s Trichrome Green Protocol

Masson’s Trichrome Green Protocol

1. De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
2. De-paraffin slides in xylene (2)——————- 2 minutes (re-use)
3. Clear slides in 100% alcohol———————– 2 minutes
4. Clear slides in 100% alcohol———————– 2 minutes
5. Hydrate slides in 95% alcohol———————- 2 minutes
6. Hydrate slides in running water——————– 5 minutes
7. Bouin’s Fixative (60 Celsius)———————- 30 minutes (re-use)
8. Wash in running water—————————— 5 minutes
9. Weigerts——————————————- 10 minutes (dump)
10. Wash in running water—————————— 5 minutes
11. Biebrich scarlet acid fuchsin———————- 5 minutes (re-use)
12. Rinse quickly————————————– 30-60 seconds
13. Phostung-Phosmoly acid (PTA-PMA)——————- 5 minutes (re-use)
14. No rinse
15. 2.5% Fast green———————————— 5 minutes (re-use)
16. Rinse quickly ————————————- 30 seconds
17. 1% Glacial acetic acid—————————– 2 minutes (re-use)
18. Dehydrate in 95% alcohol————————— 30 seconds
19. Dehydrate in 100% alcohol————————– 1 minute
20. Dehydrate in 100% alcohol————————– 2 minutes
21. Dehydrate in 100% alcohol————————– 2 minutes
22. Clear in xylene (3)——————————– 2 minutes (re-use)
23. Clear in xylene (4)——————————– 5 minutes (re-use)

Results:
Nuclei——————— Black to purple
Cytoplasm- —————- Shades of pink, red
Erythrocytes and myelin—- Scarlet
Collagen fibers———— Strong bluish-green
Mucus———————- Lighter green

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Masson’s Trichrome Blue Protocol

Masson’ Trichrome Blue
Safety equipment: Work under a hood with lab coat, gloves, and glasses.
1. De-paraffin slides in xylene (1)———————- 2 minutes (re-use)
2. De-paraffin slides in xylene (2)———————- 2 minutes (re-use)
3. Clear slides in 100% alcohol————————– 2 minutes
4. Clear slides in 100% alcohol————————– 2 minutes
5. Hydrate slides in 95% alcohol————————- 2 minutes
6. Hydrate slides in running water———————– 5 minutes
7. Bouins at 60 degrees Celsius————————– 1 hour (re-use)
8. Wash in running water ——————————– 10 minutes
9. Weigert’s Hematoxylin (make fresh) —————— 10 minutes (dump)
10. Wash in running water ——————————– 10 minutes
11. Biebrich Scarlet Acid Fuchsin————————- 10 minutes (re-use)
12. Rinse in running water——————————– 2 minutes
13. Phosphomolybdic / phosphotungstic acid—————- 10 minutes (re-use)
14. No rinse, goes directly into blue
15. Analine Blue—————————————— 10 minutes (re-use)
16. Rinse in running water——————————– 1-2 minutes
17. 1% Glacial Acetic Acid——————————– 5 minutes (re-use)
18. Dehydrate in 95% alcohol—————————— 1 minute
19. Dehydrate in 95% alcohol—————————— 1 minute
20. Dehydrate in 100% alcohol—————————– 1 minute
21. Dehydrate in 100% alcohol—————————– 2 minutes
22. Dehydrate in 100% alcohol—————————– 2 minutes
23. Clear in xylene (3)———————————– 2 minutes (re-use)
24. Clear in xylene (4)———————————– 5 minutes (re-use)

Results:
Nuclei- Black
Cytoplasm, keratin, muscle fiber and intercellular fibers- Red
Collagen- Blue