Cresyl Violet Acetate 2.5%, Aqueous

SOLUTION:

Additionally Needed:

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

APPLICATION:

Newcomer Supply Cresyl Violet Acetate 2.5%, Aqueous is a metachromatic basic dye stock solution for the demonstration of Nissl substance and nuclei in nerve tissue.  As a working solution, Cresyl Violet Acetate can be utilized in the Luxol Fast Blue (LFB) procedure or used solely as a stand-alone nerve tissue stain.

Cresyl Violet Acetate is also known as Cresyl Violet, Cresyl Fast Violet Acetate and Cresyl Echt Violet Acetate.  Due to characteristics of the dye, the actual percentage of dye concentration in Cresyl Violet powder may vary between lots.  The minimum acceptable dye content for Cresyl Violet certification is noted as 65%.

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)

Technique:  Paraffin sections cut at 8-10 microns on adhesive slides

• • • Air-dry a minimum of 30 minutes

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

PRESTAINING PREPARATION:

1. 1. If necessary, heat dry tissue sections/slides in oven.
   2. Prepare Acetic Acid 10%, Aqueous; combine and mix well.
      1. 1. Acetic Acid, Glacial, ACS (Part 10010) 10 ml
         2. Distilled Water                90 ml
         3. Store at room temperature for up to 1 year.
   3. Prepare Working Cresyl Violet Acetate 0.25%; mix well and filter.
      1. 1. Cresyl Violet Acetate 2.5%, Aqueous 5 ml
         2. Distilled Water              45 ml
         3. Acetic Acid 10%, Aqueous                  7 drops
         4. Heat filtered solution in 57°C oven; hold for Step #7.
         5. See Procedure Notes #3 and #4.

STAINING PROCEDURES:

1. 4. 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.
      1. 1. LFB procedure: stop at 95% ethyl alcohol, no distilled water rinse.
         2. Cresyl Violet Acetate stand-alone staining: continue with distilled water rinse. Proceed to Step #6.
         3. See Procedure Notes #1 and #2.
   5. LFB Stain: proceed with LFB Stain Set (Part 12218) protocol through final differentiation step and rinse thoroughly in distilled water.
   6. Continue with LFB Cresyl Violet Acetate counterstain or Cresyl Violet Acetate stand-alone staining.
   7. Stain in filtered, heated Working Cresyl Violet Acetate 0.25% (Step #3) for 6 minutes in 57°C oven.
   8. Rinse in distilled water.
   9. Dehydrate quickly to maintain Cresyl Violet Acetate stain 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. Due to possible dye percentage variance between lots of Cresyl Violet powder, a solution stronger than 0.25% Working Cresyl Violet Acetate may be necessary for optimal staining results.
   4. For enhanced staining of Working Cresyl Violet Acetate, adjust pH to 4.0 with Acetic Acid 10%, Aqueous.
   5. If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.

REFERENCES:

1. 1. Bancroft, John D. and Marilyn Gamble. Theory and Practice of Histological Techniques. 6th ed. Oxford: Churchill Livingstone Elsevier, 2008. 366-367.
   2. Carson, Freida L. and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 191-193, 207-208.
   3. Horobin, Richard and John Kiernan. Conn’s Biological Stains: A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine. 10th ed. Oxford: BIOS, 2002. 281-282.
   4. Modifications developed by Newcomer Supply Laboratory.