Sulfated Alcian Blue (SAB) Stain

SOLUTIONS:                                                                                                                 

Additionally Needed:

For storage requirements and expiration date refer to individual bottle labels. 

APPLICATION:

Newcomer Supply Sulfated Alcian Blue (SAB) Stain for Amyloid assists in identifying the extraneous protein deposits of amyloidosis, specifically in myocardial and renal biopsy specimens.

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)
Technique:  Paraffin sections cut at 8-10 microns
Solutions:  All solutions manufactured by Newcomer Supply Inc.

All Newcomer Supply stain procedures are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure.

PRESTAINING PREPARATION:

1. 1. Prepare the following three fresh solutions:
      1. 1. SAB Staining Solution

 Alcian Blue Stain, Alcoholic                               22 ml

 Sodium Sulfate 1%, Aqueous                           22 ml

 Acetic Acid, Glacial, ACS (Part 10010)                      5 ml

Allow to stand for 30 minutes before use.

1. 1. 1. 2. Acetic Acid/Alcohol Solution

 Alcohol, Ethyl Denatured, 95% (Part 10842)            44 ml

 Distilled Water                                                 44 ml

 Acetic Acid, Glacial, ACS                                 10 ml

1. 1. 1. 3. Picric Acid 2%, Alcoholic Working Solution

 Alcohol, Ethyl Denatured, 80% (Part 10843)           30 ml

Picric Acid, Saturated Alcoholic (Part 1337)             10 ml

STAINING PROCEDURE: 

1. 2. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each. Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with distilled water.
      1. 1. See Procedure Notes #1 and #2.
   3. Place in Acetic Acid/Alcohol Solution (Step #1b) for 2 minutes.
   4. Place slides in SAB Staining Solution (Step #1a) for 2 hours.
   5. Transfer directly to fresh Acetic Acid/Alcohol Solution (Step #1b) for 2 minutes.
   6. Wash well in tap water; rinse in distilled water.
   7. Alkalinize slides in Borax (Sodium Borate) 5%, Saturated Alcoholic (Part 1019) for 30 minutes.
   8. Wash well in tap water; rinse in distilled water.
   9. Prepare Hematoxylin Stain, Weigert Iron (Part 1409); combine, mix well.
      1. 1. Solution A: Ferric Chloride, Acidified   20 ml
         2. Solution B: Hematoxylin 1%, Alcoholic    20 ml

1. 10. Stain in Hematoxylin Stain, Weigert Iron for 5 minutes.
   11. Wash in tap water for 10 minutes; rinse in distilled water.
   12. Differentiate each slide individually; dip in Picric Acid 2%, Alcoholic Working Solution (Step #1c) for 20 seconds.
   13. Rinse briefly in tap water; 2-3 dips.
   14. Counterstain with Van Gieson Stain (Part 1404) for 3 minutes.
   15. Dehydrate quickly in two changes each of 95% and 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

RESULTS:

Note: Green staining tissues other than amyloid are morphologically distinguishable.

1. 1. Mast cells stain dense bright green, with typical cytoplasmic granularity.
   2. Red blood cells occasionally appear very pale green.
   3. Calcium deposits stain a dirty blue/green.

PROCEDURE NOTES:

1. 1. Drain slides after each step to prevent solution carry over.
   2. Do not allow sections to dry out at any point during procedure.
   3. If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.

REFERENCES:

1. 1. Lendrum, A. C., W. Slidders and D. S. Fraser. “Renal Hyalin: A Study of Amyloidosis and Diabetic Fibrinous Vasculosis with New Staining Methods.” Journal of Clinical Pathology 25 (1972): 373-96.
   2. Pomerance, Ariela, Gerard Slavin and Josephine McWatt. “Experience with the Sodium Sulphate-Alcian Blue Stain for Amyloid in Cardiac Pathology.” Journal of Clinical Pathology, 29 (1976): 22-26.
   3. Modifications developed by Newcomer Supply Laboratory.