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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

 

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Congo Red Stain Protocol For Amyloid

Run a control slide.                                     Control tissue – Tissue with amyloid

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. 1% Congo red ————————————— 60 minutes (re-use)
  8. Rinse in water ————————————— 1 minute
  9. Alkaline Alcohol ————————————- until background clear (re-use)
  10. Wash in running water —————————- 5 minutes
  11. Harris Hematoxylin ——————————— 30 seconds (re-use)
  12. Wash in running water —————————- 10 minutes
  13. Dehydrate in 95% alcohol———————— 30 seconds
  14. Dehydrate in 100% alcohol——————— 1 minute
  15. Dehydrate in 100% alcohol——————— 2 minutes
  16. Dehydrate in 100% alcohol——————— 2 minutes
  17. Clear in xylene (3)——————————– 2 minutes (re-use)
  18. Clear in xylene (4)——————————– 5 minutes (re-use)
  19. Coverslip

Results:         Amyloid – Pink to red                     Nuclei – Blue

With polarizing microscope

Amyloid – Green Birefringence

 

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Alizarin Red S Stain Protocol for Calcium

Run a control slide. Control tissue: Bone or tissue with calcium present

Safety equipment: Work under a hood with lab coat, gloves, and safety 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 50% alcohol——————– 1 minute
  7.   Rinse rapidly in DH2O—————————— 30 seconds
  8.   Cover the sections with alizarin Red S solution (re-use)
  9.   Observe the reaction under a microscope and remove when the red-orange lake forms ( 5 minutes to 15 mintues). The lake should be heavy but not too diffuse.

10.    Shake off excess dye and blot carefully.

11.    Dehydrate in acetone for 10 to 20 seconds (dump)

12.    Dehydrate in acetone-xylene (50:50)———–10 to 20 seconds (dump)

13.    Clear in xylene—————————————- 5 minutes (re-use)

14.    Coverslip

Results:

Calcium Deposits (except oxalate) ———————Orange-Red

This precipitate is birefringent.

 

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Hematoxylin & Eosin (H&E) Protocol

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. Stain slides in Harris Hematoxylin————–  10 minutes (re-use)
  8. Rinse slides in running water———————  2 minutes
  9. Differentiate in acid alcohol———————-   1 quick dip (re-use)
  10. Rinse in water————————————–    3 dips
  11. Dip in ammonia water—————————-    15-30 seconds (re-use)
  12. Wash in running tap water———————–    10 minutes
  13. Place in 95% alcohol——————————    2 minutes
  14. Stain in eosin—————————————    1 minute (re-use)
  15. Dehydrate in 95% alcohol————————   1 minute
  16. Dehydrate in 100% alcohol———————-   1 minute
  17. Dehydrate in 100% alcohol———————-   2 minutes
  18. Dehydrate in 100% alcohol———————-   2 minutes
  19. Clear in xylene (3)———————————   2 minutes (re-use)
  20. Clear in xylene (4)———————————   5 minutes (re-use)

Results: Nuclei –Blue   Cytoplasm – Pink     Blood – Red