Phosphotungstic Acid Hematoxylin (PTAH) Stain

(use: Demonstrates muscle striations.)

This stain formula is twice as strong as the original Mallory formulation.

Product Options:

Part # 1334A 500 ml $69.60
Part # 1334B 1 L $123.90

SOLUTION: 500 ml 1 Liter
Phosphotungstic Acid Hematoxylin (PTAH) Stain Part 1334A Part 1334B

Additionally Needed:

Phosphotungstic Acid Hematoxylin (PTAH) Control Slides Part 4565
Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842
Zenker Fixative, Modified, Zinc Chloride Part 1461
Acetic Acid, Glacial, ACS Part 10010
Potassium Permanganate 0.25%, Aqueous Part 133931
Oxalic Acid 5%, Aqueous Part 1293
Coplin Jar, Plastic Part 5184 (for microwave modification)

 

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

 

APPLICATION:

The Newcomer Supply Phosphotungstic Acid Hematoxylin (PTAH), Stain procedure, with included microwave modification, is used for the demonstration of collagen, muscle striations and central nervous system (CNS) structures.

 

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. If necessary, heat dry tissue sections/slides in oven.
  2. Prepare Zenker Fixative Working Solution; combine and mix well.

        Zenker Fixative, Modified, Zinc Chloride (Part 1461)               38 ml

        Acetic Acid, Glacial, ACS (Part 10010)                                     2 ml

 

STAINING PROCEDURE:

  1. 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. See Procedure Notes #1 and #2.
  2. Fix in Zenker Fixative Working Solution (Step #2) at 56°C for 3 hours.

        Microwave Modification:  See Procedure Note #3.

  1. Place slides in a plastic Coplin jar containing prepared Zenker Fixative Working Solution and microwave for 5 minutes at 60°C.
  1. Wash well in three changes of tap water; rinse in distilled water.
  2. Place in Potassium Permanganate 0.25%, Aqueous (Part 133931) for 10 minutes.
  3. Wash in three changes of tap water; rinse in distilled water.
  4. Place in Oxalic Acid 5%, Aqueous (Part 1293) for 10 minutes.
  5. Wash in three changes of tap water; rinse in distilled water.
  6. Place in PTAH Stain for 12-24 hours at room temperature, or 2 hours at 56°C.
    1. See Procedure Note #4.

        Microwave Modification:

  1. Place slides in a plastic Coplin jar containing PTAH Stain and microwave for 7 minutes at 70°C.
  1. 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.
    1. Dehydrate quickly as alcohol may extract stain from sections.

 

RESULTS:

Collagen, cartilage, elastic fibers Deep reddish brown
Muscle striations, fibrin, keratin Dark blue
Glia fibers Dark blue
Myelin Lighter blue
Neurons Salmon/pink
Nuclei Blue

 

PROCEDURE NOTES:

  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 suggested microwave procedure has been tested at Newcomer Supply.  This procedure is a guideline and techniques should be developed for use in your laboratory.
  4. Newcomer Supply PTAH Stain formula is twice as strong as the original Mallory formulation; adjust staining time according to preference of intensity.  Suggested staining time at 37°C is 18 hours.
  5. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

 

REFERENCES:

  1. Bancroft, John D., and Marilyn Gamble. Theory and Practice of Histological Techniques. 6th ed. Oxford: Churchill Livingstone Elsevier, 2008.130-131.
  2. Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-Instructional Text. 4th ed. Chicago: ASCP Press, 2015. 178-180, 201-202.
  3. Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 193-194.
  4. Modifications developed by Newcomer Supply Laboratory.