Solutions and Reagents:

Eosin-Phloxine B Solution:

      Eosin Stock Solution:

      Eosin Y ---------------------------------- 1 g

      Distilled water ------------------------- 100 ml

      Mix to dissolve.

      Phloxine Stock Solution:

      Phloxine B ------------------------------ 1 g

      Distilled water ------------------------- 100 ml

      Mix to dissolve.

      Eosin-Phloxine B Working Solution:

      Eosin stock solution ------------------ 100 ml

      Phloxine stock solution -------------- 10 ml

      Ethanol (95%) ------------------------- 780 ml

      Glacial acetic acid --------------------- 4 ml

      Mix well.

Alternate for Eosin-Phloxine B Solution

Eosin Y Solution:

   Eosin Y Stock Solution (1%):

   Eosin Y --------------------------------------- 10 g

   Distilled water ------------------------------- 200 ml

   95% Ethanol ---------------------------------- 800 ml

   Mix to dissolve and store at room temperature.

   Eosin Y Working Solution (0.25%):

   Eosin Y stock solution -------------------- 250 ml

   80% Ethanol -------------------------------- 750 ml

   Glacial acetic acid (concentrated) ------- 5 ml

   Mix well and store at room temperature.

Hematoxylin Solution (Mayer):

      Potassium or ammonium (alum) -------- 50 g

      Hematoxylin ------------------------------- 1 g

      Sodium iodate ----------------------------- 0.2 g

      Citric acid ---------------------------------- 1 g

      Distilled water ----------------------------- 1000 ml

      Stir to dissolve the chemicals in the order listed above. For example, dissolve alum in 1000 ml distilled water first. When alum is completely dissolved, add hematoxylin. When hematoxylin is completely dissolved, add sodium iodate, etc.

Procedure:

1.    Deparaffinize sections, 2 changes of xylene, 10 minutes each.

2.    Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.

3.    95% alcohol for 2 minutes and 70% alcohol for 2 miuntes.

4.    Wash briefly in distilled water.

5.    Stain in Mayer hematoxylin solution for 8 minutes.

6.    Wash in warm running tap water for 10 minutes.

7.    Rinse in distilled water.

8.    Rinse in 95% alcohol, 10 dips.

9.    Counterstain in eosin-phloxine B solution (or eosin Y solution) for 30 seconds to 1 minute.

10.  Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each.

11.  Clear in 2 changes of xylene, 5 minutes each.

12.  Mount with xylene based mounting medium.

Results:

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

Cytoplasm ---------------------------------- pink to red