FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

0.25% Potassium Permanganate Solution

Potassium permanganate -------------------------------------------------------                0.25 gm

Distilled water -----------------------------------------------------------------                100.00 ml

1% Oxalic Acid Solution

Oxalic acid ---------------------------------------------------------------------                1.0 gm

Distilled water -----------------------------------------------------------------                100.0 ml

Modified PTAH Solution

Solution A

Dissolve 0.5 gm of hematoxylin (C.I. 75290) in 400 ml of distilled water with the aid of gentle heat.  The temperature of the solution should not exceed 60o C.  Allow to cool for 20 minutes and add 0.089 gm of potassium permanganate.  Allow to stand for one hour.

Solution B

Dissolve 10 gm of phosphotungstic acid in 100 ml of distilled water.

PTAH Staining Solution

Combine solutions A and B and let stand for one hour.  Store in an amber bottle away from strong light.  The solution is stable for at least several months.

• Use control slide.

1. Deparaffinize and hydrate slides to distilled water.
2. Potassium permanganate solution for 5 minutes.
3. Rinse in three changes of distilled water.
4. Bleach in oxalic acid solution for 2 minutes.
5. Wash in running water for 1 minute and rinse with two changes of distilled water.
6. Mordant in 40 ml of Zenker’s solution that contains 5% acetic acid in a glass Coplin jar and microwave at power level 1 (60W) for 1 minute.     Transfer to a 37o C oven for 2 ½ hours.  An alternate method is to place the slides in Zenker’s solution overnight at room temperature.
7. Wash in running water 3 minutes.
8. Two changes of distilled water.
9. Place in 40 ml of PTAH solution in a glass Coplin jar and microwave at power level 1 (60W) for 1 minute.  Transfer to a 37o C oven for 3 hours.  An alternate method is to place the slides in PTAH solution overnight at room temperature.
10. Transfer slides directly to 95% alcohol in Coplin jars, three changes.
11. Dehydrate with absolute alcohol, four changes.
12. Clear in xylene, three or four changes.
13. Mount with synthetic resin.

Nuclei -------------------------------------------------------------        blue

Skeletal and cardiac muscle ----------------------------------------        blue, with cross striations well defined

Fibrin ----------------------------------------------------------------        blue

Fibroglia and microglia ---------------------------------------------        blue

Collagen ------------------------------------------------------------                reddish

Erythrocytes --------------------------------------------------------        greyish blue

Glial fibers ----------------------------------------------------------        blue

Myelin ---------------------------------------------------------------        blue

Some authors recommend that the tissue sections be treated with potassium permanganate and oxalic acid after they have been mordanted in acidified Zenker’s.  We have found that this sequence tends to give less satisfactory results in demonstrating cross striations of muscle than the sequence described in this method.

This stain is most frequently used to demonstrate fibrin. It is occasionally used to demonstrate skeletal muscle cells (cross striations) in mesenchymal tumors, and the degree of skeletal muscle degenerative changes in various myopathies. In the central nervous system it is useful for staining glial fibers.

Mallory, F.B.:  Pathological Technique, New York, Hafner, 1968, pp. 76-77.

Meloan, S.N. and Puchtler, H:  On the chemistry of phosphotungstic acid-hematein: development of a rapidly ripening PTAH solution.  J. Histotechnol. 11:153-157, 1988.