Trichrome Stain, Gomori One-Step, Light Green

Tech Memo 1: Trichrome Stain, Gomori One-Step, Light Green

SOLUTION:

Additionally Needed:

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

APPLICATION:

Newcomer Supply Trichrome Stain, Gomori One-Step, Light Green procedure, with included microwave modification, uses a one-step solution combining a plasma stain and a connective tissue stain to differentially demonstrate collagen and muscle fibers.

METHOD:

Fixation: Formalin 10%, Phosphate Buffered (Part 1090)
Technique:  Paraffin sections cut at 4 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. If necessary, heat dry tissue sections/slides in oven.
   2. Preheat 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
   7. Stain slides 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. Stain in Trichrome Stain, Gomori One-Step, Light Green for 20 minutes.
   10. Differentiate in Acetic Acid 0.5%, Aqueous (Part 100121); 2 minutes.
   11. Rinse quickly in distilled water.
   12. 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:

1. 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. 165-166.
   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.

Additionally Needed:

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

 
APPLICATION:

Newcomer Supply Trichrome Stain, Gomori One-Step, Light Green for frozen muscle biopsies uses a one-step solution combining a plasma stain and connective tissue stain.  This procedure provides excellent staining results on fresh non-fixed frozen muscle biopsy sections for  demonstration of muscle fiber morphology and surrounding connective tissue.

METHOD:

Technique: Frozen muscle sections cut at 8 microns on adhesive slides or coverglass

1. 1. 1. See Procedure Note #1.

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

PRESTAINING PREPARATION:

1. 1. Allow Trichrome Stain, Gomori One-Step, Light Green to reach room temperature prior to use.
   2. Prepare Acetic Acid 0.25%, Aqueous; combine and mix well.
      1. 1. Acetic Acid 0.5%, Aqueous (Part 100121) 20 ml  
         2. Distilled Water                                        20 ml

STAINING PROCEDURE:

1. 3. Air-dry frozen sections a minimum of 10 minutes prior to staining.
      1. 1. See Procedure Note #2.
   4. Stain air-dried frozen muscle sections in Hematoxylin Stain, Harris Modified (Part 1201) or Hematoxylin Stain, Harris (Part 12013) for 5 minutes.
   5. Rinse in running tap water for 3 minutes.
      1. 1. Do not differentiate or use a bluing agent.
   6. Stain in Trichrome Stain, Gomori One-Step, Light Green for 18-20 minutes in a 38°- 40°C oven.
   7. Differentiate in Acetic Acid 0.25%, Aqueous (Step #2); 1-2 dips.
   8. 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. For optimal results and minimal tissue artifact, fresh non-fixed muscle biopsies should be expediently snap frozen using an isopentane (2-Methylbutane) – liquid nitrogen freezing method.
   2. Do not fix sections or use a Bouin Fluid mordant prior to staining.
      1. 1. Exposure to fixative or mordant will alter staining results.
   3. If using a xylene substitute, follow manufacturer’s recommendation for clearing step.

REFERENCES:

1. 1. Carson, Freida L. and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 328-329.
   2. Dubowitz, Victor and Caroline A. Sewry. Muscle Biopsy: A Practical Approach. 2nd ed. London: Baillière, 1985.30.
   3. Mitchell, Jean and Andrew Waclawik. “Muscle Biopsy in Diagnosis of Neuromuscular Disorders: The Technical Aspects, Clinical Utility, and Recent Advances.” The Journal of Histotechnology 30.4 (2007): 257-269.
   4. Sheehan, Dezna C. and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 191-192.
   5. Modifications developed by Newcomer Supply Laboratory.