Dissolve the hematein in 200 mL of the water and the phosphotungstic acid in the rest. Combine the solutions. Keep in a bottle with a tightly fitting cap to ensure atmospheric oxygen is excluded. The stain may be used after a day or two.
Place into the PTAH solution for 12 - 24 hours at room temperature.
Rinse well with water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Nuclei, erythrocytes, fresh fibrin, muscle striations – blue
Background – red
If formalin fixed, the sections may be treated with acid dichromate for 30 minutes.
Potassium dichromate, 3% aqueous
10% hydrochloric acid in absolute ethanol.
The staining may be done in an oven at about 60°C for a few hours, but the results are often less satisfactory.
Many substances have been stated to be stained blue. Sometimes the red staining overshadows blue staining, and the section may need to be washed with water to remove excess red. Excess blue may be removed by extending the treatment with ethanol during dehydration.
PTAH may also be made with hematoxylin and oxidised as any other hematoxylin solution. Atmospheric oxidation is often recommended, but takes a few months. Place the solution in a flask with a loose cotton batting stopper to facilitate exposure to air. Test periodically and, when staining is satisfactory, place into a bottle with a tightly fitting lid to inhibit atmospheric oxidation. Alternatively, the hematoxylin may be chemically oxidised. This is usually done with 12 mL of 1% aqueous potassium permanganate. The solution may be used after a day or two. Sodium iodate can also be used, in which case half the hematoxylin will be oxidised by 0.1 grams sodium iodate. Bring to the boil, cool and use immediately, or leave a few days at room temperature.
Reference Bancroft, J. D. and Stevens, A., Theory and Practice of Histological Techniques, Ed. 2,
Churchill Livingstone, London, UK.