FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 4-6 µm.

SOLUTIONS:

Modified Lillie-Mayer Hematoxylin

Hematoxylin, C.I. 75290 --------------------------------------------------------                5.0 gm

Ammonium alum (aluminum ammonium sulfate) ----------------------------------                50.0 gm

Distilled water -------------------------------------------------------------------        800.0 ml

Glycerol -------------------------------------------------------------------------        200.0 ml

Sodium iodate -------------------------------------------------------------------        0.3 gm

       Dissolve the hematoxylin and ammonium alum in the distilled water. 

       Then add the glycerol and sodium iodate.  Before use add 3 ml of

       acetic acid per 100 ml of solution.  This increases the precision and

       selectivity of the nuclear stain.  Filter before use.

0.3% Sodium Borate

(See PAS Method)

1% Alcoholic Eosin Solution (Stock)

Eosin Y, C.I. 45380 -------------------------------------------------------------        1.0 gm

Distilled water --------------------------------------------------------------------        20.0 ml

       Dissolve and add:

Alcohol, 95% ---------------------------------------------------------------------        80.0 ml

Eosin Solution (Working)

Eosin stock solution --------------------------------------------------------------        100.0 ml

Alcohol, 80% --------------------------------------------------------------------        300.0 ml

       Just before use add 1 ml of acetic acid to each 100 ml of stain.

Eosin-Phloxine Solution

Stock Eosin

Eosin Y, C.I. 45380 -------------------------------------------------------------        1.0 gm

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

Stock Phloxine

Phloxine B, C.I. 45410 ----------------------------------------------------------        1.0 gm

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

Working Eosin-Phloxine Solution

Stock eosin -----------------------------------------------------------------------        100.0 ml

Stock phloxine --------------------------------------------------------------------        10.0 ml

Alcohol, 95% ---------------------------------------------------------------------        780.0 ml

Acetic acid, glacial ----------------------------------------------------------------        8.0 ml

1.        Deparaffinize and hydrate to distilled water.

2.        Modified Lillie-Mayer hematoxylin for 1 to 3 minutes.

3.        Blue hematoxylin in 0.3% sodium borate for 15 seconds.

4.        Rinse in four changes of distilled water.

5.        Place in 95% alcohol for 30 seconds.

6.        Counterstain with working alcoholic eosin or working eosin-phloxine for 30 seconds to 2 minutes.  The time is determined by the age of the solutions and the depth of the counterstain desired.

7.        Dehydrate in graded alcohols.

8.        Clear in three or four changes of xylene.

9.        Mount with synthetic resin.

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

Cytoplasm -----------------------------------------------------------        various shades of pink which identifies different tissue components

Erythrocytes ---------------------------------------------------------        shades of pink to red

Alcoholic hyalin ------------------------------------------------------        pink if the eosin-phloxine counterstain is used

Lillie, R.D., and Fullmer, H.M.:  Histopathologic Technique and Practical Histochemistry, 4th ed., New York, McGraw-Hill, 1976, pp. 205-208.

Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., New York, McGraw-Hill, 1968, pp. 34-35.