FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

Patent Blue V Solution (Stock)

Patent blue V, C.I. 42045 ------------------------------------------------------                0.25 gm

Acetic acid, 2% ----------------------------------------------------------------                25.0 ml

Zinc dust, granulated -----------------------------------------------------------                2.50 gm

       Mix well on a magnetic stirrer.  The solution will become

       a pale greenish-blue.  Filter and store in a refrigerator at

       3-6o C.  The solution is stable for about one week.

Patent Blue V Solution (Working)

Patent blue V solution ----------------------------------------------------------                10.0 ml

Acetic acid ---------------------------------------------------------------------                2.0 ml

Hydrogen peroxide, 3% --------------------------------------------------------                1.0 ml

       Prepare just before use.

Nuclear Fast Red Solution

(See Pearl’s Method for Iron)

• Use control slide.

1. Deparaffinize and hydrate to distilled water.
2. Place slides on a staining rack and flood with freshly prepared patent blue V working solution for 5 minutes.
3. Rinse in three changes of distilled water.
4. Nuclear fast red for 3 minutes.
5. Rinse in three changes of distilled water.
6. Dehydrate in graded alcohols.
7. Clear in three or four changes in xylene.
8. Mount with synthetic resin.

Hemoglobin --------------------------------------------------------                blue to green

Nuclei -------------------------------------------------------------        red

According to Lillie aniline blue (C.I. 42755) may be substituted for patent blue V in this procedure.  He states that it will decolorize to a light transparent green with zinc and hot dilute acetic acid and will stain hemoglobin blue.  We tried two different samples of aniline blue in this procedure and neither one gave satisfactory staining results.

Lison indicated in this method that the patent blue V stock solution should be boiled until it becomes nearly colorless.  We found that the solution becomes nearly colorless without the need to boil it.

Lillie, R.D. and Fullmer, H.M.:  Histopathologic Technic and Practical Histochemistry, 4th ed., New York, McGraw-Hill, 1976, pp. 447.