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Mallory PTAH Stain Protocol For Muscle Striations

This is an old stain not used much anymore for diagnosis.  It has been replaced by immunohistochemisty.

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. If sections are formalin fixed, mordant in Zenker fixative containing 5% acetic acid overnight at room temperature.
  8. Rinse in running water ————————— 2 minutes
  9. Place in Gram’s Iodine ————————— 15 minutes

10. Rinse in running water ————————— 1 minute

11. Place in 5% sodium thiosulfate ——————– 3 minutes

12. Wash in running water ————————— 10 minutes

13. Place in 0.25% potassium permanganate ——— 5 minutes

14. Rinse in running water ————————— 1 minute

15. Place in 5% oxalic acid ————————— 1 minute

16. Stain in PTAH at room temp ——————— Overnight

17. Dehydrate in 95% alcohol————————- 30 seconds

18. Dehydrate in 100% alcohol———————– 30 seconds

19. Dehydrate in 100% alcohol———————- 30 seconds

20. Dehydrate in 100% alcohol———————- 30 seconds

21. Clear in xylene (3)——————————- 2 minutes (re-use)

22. Clear in xylene (4)——————————- 5 minutes (re-use)

Results:

Cross-striations, fibrin – Blue

Nuclei – Blue

Collagen – Red-Brown

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Bielschowsky for Neurofibrils and Senile Plaques (Alzheimer)

In addition to the Tau and Amyloid proteins discovered through IHC, this silver stain indicates Alzheimer disease.

Control tissue: Brain Cortex, Spinal Cord

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. 10% Silver nitrate ——————————  30 minutes in 40C oven
  8. Place slides in DH2O—————————-  until new solution is made
  9. Slowly add ammonium hydroxide to the 10% silver nitrate until it is clear.
  10. Place slides in solution in 40C oven ————– 30 minutes
  11. Place in developer solution ———————- 5 minutes (re-use)
  12. Slides should be golden, use scope to check.
  13. Place in 1% ammonium hydroxide to stop reaction – 1 minute
  14. Wash in water ———————————– 2-5 minutes
  15. Place in 5% sodium thiosulfate ——————- 5 minutes (re-use)
  16. Wash in running water ————————–  5 minutes
  17. Dehydrate in 95% alcohol———————— 30 seconds
  18. Dehydrate in 100% alcohol———————–  1 minute
  19. Dehydrate in 100% alcohol———————–  2 minutes
  20. Dehydrate in 100% alcohol———————–  2 minutes
  21. Clear in xylene (3)——————————-  2 minutes (re-use)
  22. Clear in xylene (4)——————————-  5 minutes (re-use)

Results:           Neurofibrils and senile plaques- black

Background- yellow to brown

 

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Grocott’s Methanamine Silver (GMS)

Here is another fungus stain.  Some of the fungi that you might stain with PAS but do not will stain with the GMS.  The silver used must be disposed of properly (not down the sink).  When using gold chloride remember to use plastic forceps, metal forceps will corrode on contact.  All glassware should be chemically cleaned before use to reduce contamination.  Pictures will be coming soon.

Use Control Tissue with known fungi.

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. Place in 10% chromic acid ———————- 15 minutes (re-use)
  8. Rinse in running water ————————- 1 minute
  9. Place in 1% sodium bisulfate ——————– 1 minute (re-use)
  10. Rinse in running water ————————- 1 minute
  11. Place in working Methanamine silver at 60C—- 60 minutes
  12. Rinse in running water ————————- 1 minute
  13. Place in 0.1% gold chloride ——————— 4 minutes (re-use)
  14. Rinse in running water ———————— 1 minute
  15. Place in 2% sodium thiosulfate —————- 4 minutes (re-use)
  16. Rinse in running water ———————–  1 minute
  17. Place in working light green ——————  30 seconds
  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)
  1. 23.   Clear in xylene (4)——————————– 5 minutes (re-use)

Results:         Fungi – Sharply delineated in Black      

Mucin – Taupe to dark Grey

                       Inner parts of mycelia and hyphae – Old Rose            

Background – Pale Green

 

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Safranin O Fast green Stain Protocol

Anyone out there doing articular cartilage studies?  This stain is for you.  A fast easy stain that can give beautiful results.  I wish all stains were this easy.  Take a look.

Control tissue:  Use bone with joint attached.

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. Harris Hematoxylin—————————-  5 minutes (re-use)
  8. Define in 1% acid alcohol———————— 1 dip (re-use)
  9. Blue in ammonium water———————–  30 seconds (re-use)
  10. 10.  Rinse in water—————————— 1 minute
  11. Stain in Fast green—————————— 5 minutes (re-use)
  12. Rinse in water———————————-  30 seconds
  13. 1% acetic acid———————————– 30 seconds (re-use)
  14. Stain in Safranin O—————————— 15 minutes (re-use)
  15. Rinse in water———————————- 30 seconds
  16. Dehydrate in 95% alcohol———————– 30 seconds
  17. Dehydrate in 100% alcohol———————- 1 minute
  18. Dehydrate in 100% alcohol———————- 2 minutes
  19. Dehydrate in 100% alcohol———————- 2 minutes
  20. Clear in xylene (3)—————————— 2 minutes (re-use)
  21. Clear in xylene (4)—————————— 5 minutes (re-use)
  22. Coverslip

Results:

Articular cartilage (proteoglycan) – Red

Nuclei- Blue

Background- Green

 

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