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Special Stain Solution Protocols Page

Hello,  We have starting adding our solution protocols on a new page.  These are all the protocols that we use now. A professional histology service.

http://histologistics.com/histology/special-stain-solution-protocols/

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Periodic Acid Schiff (PAS) Stain 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. 0.5% periodic acid ——————————– 5 minutes (re-use)
  8. Schiff’s reagent at 37C—————————- 15 minutes (re-use)
  9. Wash in running water ————————— 10 minutes
  10. If staining for glycogen, Hematoxylin ——— 20 seconds (re-use)
  11. If staining for fungus, light green ————— 30 seconds (re-use)
  12. Dehydrate in 95% alcohol———————— 30 seconds
  13. Dehydrate in 100% alcohol———————- 1 minute
  14. Dehydrate in 100% alcohol———————- 2 minutes
  15. Dehydrate in 100% alcohol———————- 2 minutes
  16. Clear in xylene (3)——————————— 2 minutes (re-use)
  17. Clear in xylene (4)——————————— 5 minutes (re-use)

Results:           Hematoxylin – Glycogen, mucin, fibrin, or thrombi, colloid droplets, hyaline of arteriosclerosis, hyaline deposits in glomeruli, granular cells in the renal arterioles where preserved, most basement membranes, colloid of pituitary stalks and thyroid, amyloid infiltration may show a positive reaction – Rose to Purplish Red

                        Nuclei – Blue

Results:          Light Green – Fungi – Red                 Background – Pale Green

 

 

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Alcian Blue / PAS Stain Protocol

Here is the protocol for alcian blue periodic acid schiff  (AB /PAS).  At the bottom of the post is the link for printing.

Control tissue: Large Intestine

Rat colon PASAB2

  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 slides in 3% Acetic Acid————- 3 minutes (re-use)
  8. Alcian Blue ———————————– 30 minutes (re-use)
  9. Wash in running water ——————— 10 minutes
  10. Oxidize in 0.5% periodic acid ————- 10 minutes (re-use)
  11. Wash in running water ——————— 5 minutes
  12. Schiff reagent (room temp)—————- 20 minutes (re-use)
  13. Wash in running water ——————— 10 minutes
  14. Dehydrate in 95% alcohol—————— 30 seconds
  15. Dehydrate in 100% alcohol—————- 1 minute
  16. Dehydrate in 100% alcohol—————- 2 minutes
  17. Dehydrate in 100% alcohol—————- 2 minutes
  18. Clear in xylene (3)————————— 2 minutes (re-use)
  19. Clear in xylene (4)————————— 5 minutes (re-use)

Results: Polysaccharides and Mucosubstances containing hexoses or deoxyhexoses with vicinal glycol groups – Magenta to Red

Hyaluronic acid, sialomucins and all but the most strongly acidic sulfated mucosubstances – Blue

PAS Alcian Blue

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Luxol Fast Blue Stain Variations

We did this as a test to see different versions of luxol fast blue (LFB). LFB is a stain for the myelin sheath or myelinated axons in brain and spinal cord.  We tested on rat brain.  There are 4 variations.  They are; LFB / Cresyl violet (CR), LFB /H&E, LFB / Periodic acid schiff (PAS) and LFB / nuclear fast red (NFR).   The H&E and NFR versions are for background staining only.  The PAS could show glycogen or fungus present and the CR shows nissl bodies.

Rat brain LFB / CV 20x
Rat brain LFB / CV 20x
Rat brain LFB / H&E 20x
Rat brain LFB / H&E 20x
Rat brain LFB / NFR 20x
Rat brain LFB / NFR 20x
Rat brain LFB / PAS 20x
Rat brain LFB / PAS 20x