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FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 8-12 μm.

SOLUTIONS:

0.1% Luxol Fast Blue Solution

Luxol fast blue, MBS -----------------------------------------------------------                0.1 gm

Alcohol, 95% ---------------------------------------------------------------------        100.0 ml

Acetic acid, 10% -----------------------------------------------------------------        0.5 ml

0.05% Lithium Carbonate Solution

Lithium carbonate ---------------------------------------------------------------                0.05 gm

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

70% Alcohol Solution

Alcohol, 100% -------------------------------------------------------------------        70.0 ml

Distilled water --------------------------------------------------------------------        30.0 ml

0.5% Periodic Acid Solution

Periodic acid ----------------------------------------------------------------------        0.5 gm

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

Modified Lillies Schiff Solution

(see PAS method)

0.3% Sodium Borate

(See PAS method)

Cresyl Violet Acetate Solution

*Cresyl violet acetate -----------------------------------------------------------                0.04 gm

Distilled water --------------------------------------------------------------------        40.00 ml

Oxalic acid, 1% ------------------------------------------------------------------        0.20 ml

       *Cresyl echt violet may be substituted for cresyl violet acetate.

1.        Deparaffinize and hydrate to 95% alcohol.

2.        Place in 40 ml of luxol fast blue solution in a glass Coplin jar and microwave at power level 1 (60W) for 3 minutes.  Allow the slides to remain in the hot solution for 30 minutes.

3.        Rinse off excess stain in 95% alcohol.

4.        Rinse in three changes of distilled water.

5.        Begin differentiation by placing in 0.05% lithium carbonate for 5 seconds.

6.        Continue differentiation in 70% alcohol for 5-10 seconds.

7.        Rinse in four changes of distilled water.  Check under microscope and repeat steps 5 and 6 if necessary.

8.        Place in 0.5% periodic acid for 10 minutes.

9.        Rinse in four changes of distilled water.

10.        Place in modified Lillies Schiff solution for 10 minutes.

11.        Rinse in three changes of distilled water.

12.        Place in 0.3% sodium borate for 15 seconds and rinse in four changes of distilled water.

13.        Cresyl violet acetate solution for 5 minutes.

14.        Rinse in three changes of distilled water.

15.        Dehydrate in graded alcohols.

16.        Clear in three or four changes of xylene.

17.        Mount with synthetic resin.

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

PAS positive elements -----------------------------------------------                rose to red

Capillaries -----------------------------------------------------------        red

Nuclei ---------------------------------------------------------------        purple-blue

The use of the microwave oven greatly reduces the time required to perform the method.  The temperature of the solution should be 75-80˚  C.  Another way of performing the stain is by staining overnight in the luxol fast blue solution in 56-60˚ C oven.

Garvey, W. et al:  Combined modified periodic acid-Schiff and batch staining method.  J. Histotechnol. 15:117-120, 1992.

Bajues, E.:  Conditioning Factors for Cardiac Necrosis, International Medical Book Corporation, 131:1963.

Luna, L.G.:  Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., 204.

McManus, J.F.A., and Mowry, R.W.:  Staining Methods Histologic and Histochemical, New York, Paul B. Hoeber, 1960, pp. 331-333.