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FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 10-12 µm.

SOLUTIONS:

Gallocyanin Solution

Chromium potassium sulfate, CrK(SO4)2 • 12H20) -----------------------------        5.00 gm

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

Gallocyanin, C.I. 51030 ---------------------------------------------------------        0.15 gm

       Heat and bring to boiling.  Boil for 15-20 minutes.  Filter when cool.

0.5% Phloxine Solution

Phloxine B, C.I. 45410 ----------------------------------------------------------        0.5 gm

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

       The solution is stable for one month.

0.05% Fast Green Solution

Fast green FCF, C.I. 42053 -----------------------------------------------------        0.05 gm

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

1.        Deparaffinize and hydrate to distilled water.

2.        Place in freshly filtered gallocyanin solution and stain overnight at room temperature.

3.        Wash in running tap water for 5 minutes and rinse in two changes of distilled water.

4.        Phloxine solution for 5 minutes.

5.        Wash in running tap water for 3 minutes.

6.        Mordant in 5% aqueous phosphotungstic acid for 1 minute.

7.        Wash in running tap water for 3 minutes.

8.        Place in 1% acetic acid for 2 minutes.

9.        Differentiate in 80% alcohol for 1-2 minutes.  Check with a microscope after 1 minute.  Myelin should stand out clearly.

10.        Place in 1% acetic acid for 1 minute.

11.        Fast green solution for 1 minute.

12.        Dehydrate in three changes of 95% alcohol (Coplin jars) and three changes of absolute alcohol.

13.        Clear in three or four changes of xylene.

14.        Mount with synthetic resin.

Myelin ---------------------------------------------------------------                magenta

Glial fibers -----------------------------------------------------------        green

Nissl plump astrocytes -----------------------------------------------        green

Nuclei ---------------------------------------------------------------        black or blue black

Nucleoli -------------------------------------------------------------        usually red

Collagen -------------------------------------------------------------        green

Neurons -------------------------------------------------------------        black

Oligodendrocytes ----------------------------------------------------        red or black

Lapham, L.W., et al.:  A New Paraffin Method for the Combined Staining of Myelin and Glial Fibers.  J. Neuropath.  Exp. Neurol. 23:156-160, 1964