Aniline Blue Stain, Aqueous

Tech Memo 1: Trichrome Stain, Masson, Aniline Blue

SOLUTION:

Additionally Needed:

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

APPLICATION:

Newcomer Supply Trichrome Stain, Masson, Aniline Blue procedure, with included microwave modification, is used to differentially demonstrate connective tissue elements, collagen and muscle fibers.

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)
Technique:  Paraffin sections cut at 4 microns
Solutions:  All solutions are 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. If necessary, heat dry tissue sections/slides in oven.
   2. Preheat in Bouin Fluid (Part 1020) to 56-60°C in oven or water bath. (Skip if using overnight method or microwave procedure.)

STAINING PROCEDURE:

1. 3. 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.
   4. Mordant in preheated Bouin Fluid (Step #2) for one hour at 56-60°C or overnight at room temperature. Cool at room temperature for 5-10 minutes.
      1. 1. Skip Step #4 if tissue was originally Bouin fixed.

       Microwave Modification:  See Procedure Note #3.

1. 1. 1. 2. Place slides in a plastic Coplin jar containing Bouin Fluid. Microwave for 5 minutes at 60°C.

1. 5. Wash well in running tap water; rinse in distilled water.
   6. Prepare fresh Weigert Iron Hematoxylin (Part 1409); combine, mix well.
      1. 1. Solution A: Ferric Chloride, Acidified 20 ml
         2. Solution B: Hematoxylin 1%, Alcoholic 20 ml

1. 7. Stain in fresh Weigert Iron Hematoxylin for 10 minutes.
   8. Wash in running tap water for 10 minutes; rinse in distilled water.
      1. 1. See Procedure Note #4.
   9. Place in Biebrich Scarlet-Acid Fuchsin Stain, Aqueous (Part 10161) for 2 minutes.
   10. Rinse in distilled water.
   11. Place in Phosphomolybdic-Phosphotungstic Acid, Aqueous (Part 1332) for 10 to15 minutes.
   12. Transfer directly into Aniline Blue Stain, Aqueous for 5 minutes.
   13. Rinse in distilled water.
   14. Place in Acetic Acid 0.5%, Aqueous (Part 100121) for 3 to 5 minutes.
   15. Dehydrate 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:

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. The microwave procedure was tested using a laboratory-grade microwave oven. This procedure is a guideline and techniques should be developed for use in your laboratory.
   4. If Weigert Iron Hematoxylin is not completely washed from tissue sections, nuclear and cytoplasmic staining may be compromised.
   5. If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.

REFERENCES:

7. 1. Brown, Richard. Histologic Preparations: Common Problems and Their Solutions. Northfield, Ill.: College of American Pathologists, 2009. 95-101.
   2. Carson, Freida L. and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 162-165.
   3. Sheehan, Dezna C. and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 191-192.
   4. Vacca, Linda L. Laboratory Manual of Histochemistry. New York: Raven Press, 1985. 308-310.
   5. Modifications developed by Newcomer Supply Laboratory.