Phosphotungstic Acid Hematoxylin (PTAH)

PRODUCT SPECIFICATIONS:

Tissue:  Positive staining striated muscle.
Fixation: Formalin 10%, Phosphate Buffered (Part 1090).
Section/Glass: Paraffin sections cut at 4 microns on Superfrost™ Plus slides.
Quality Control Stain:  PTAH quality control stained slide(s) included.
Reactivity: Guaranteed product specific reactivity for one year from date of receipt. Revalidate after one year to verify continued reactivity. 
Storage: 15-30°C in a light deprived and humidity controlled environment.
Intended Use: To verify histological techniques and reagent reactivity.

Before using unstained control slides, review the enclosed stained slide(s) to ensure that this tissue source is acceptable for testing needs.

CONTROL SLIDE VALIDATION:

APPLICATION:

The Newcomer Supply Phosphotungstic Acid Hematoxylin (PTAH) Control Slides are for the positive histochemical demonstration of muscle striations and collagen in tissue sections.

PRESTAINING PREPARATION:

1. 1. Heat dry sections in oven according to your laboratory protocol.
   2. Prepare Zenker Fixative Working Solution; combine and mix well.

1. 1. 1. Solution A: Zenker Fixative, Modified, Zinc Chloride       38 ml
      2. Solution B: Acetic Acid, Glacial, ACS                                2 ml

NEWCOMER SUPPLY VALIDATION 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. Fix in Zenker Fixative Working Solution (Step #2) at 56°C; 3 hours.

        Microwave Modification:  See Procedure Note #3.

1. 1. 1. 1. Place slides in a plastic Coplin jar containing Zenker Fixative Working Solution. Microwave 5 minutes at 60°

1. 5. Wash well in three changes of tap water; rinse in distilled water.
   6. Place in Solution C: Potassium Permanganate 0.25%, Aqueous for 10 minutes.
   7. Wash in three changes of tap water; rinse in distilled water.
   8. Place in Solution D: Oxalic Acid 5%, Aqueous for 10 minutes.
   9. Wash in three changes of tap water; rinse in distilled water.
   10. Stain in Solution E: PTAH Stain, Modified Mallory for 12-24 hours at room temperature, or 2 hours at 56°
       1. 1. See Procedure Note #4.

Microwave Modification:

1. 1. 1. 2. Place slides in a plastic Coplin jar containing Solution E: PTAH Stain, Modified Mallory. Microwave 7 minutes at 70°

1. 11. 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. 1. Dehydrate quickly, alcohol may extract stain from sections.

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. Adjust staining time according to preference of intensity. Suggested staining time at 37°C is 18 hours.
   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, 2008.130-131.
   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.