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

       Dissolve dye in alcohol.  Add 0.5 ml of 10% glacial acetic acid

       to each 100 ml.

Cresyl Violet Acetate Solution

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

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

1% oxalic acid --------------------------------------------------------------------        0.20 ml

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

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

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 solution for 5 seconds.

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

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

8.        Cresyl violet acetate solution for 5 minutes.

9.        Rinse in three changes of distilled water.

10.        Dehydrate in graded alcohols.

11.        Clear in xylene, three or four changes.

12.        Mount with synthetic resin.

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

Cell products --------------------------------------------------------        pink to violet

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 after it is microwaved.  Another way of performing the stain is by staining overnight in the luxol fast blue solution in a 56-60˚  C oven.

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., New York, McGraw-Hill, 1968, pp. 203-204.