Solutions and Reagents:
1% Acid Alcohol Solution (for differentiation):
Hydrochloric acid --------------- 1 ml
70% ethanol --------------------- 100 ml
Mix well.
0.2% Ammonia Water Solution (Bluing):
Ammonium hydroxide (concentrated) ----- 2 ml
Distilled water ------------------------------- 1000 ml
Mix well.
Lithium Carbonate Solution (Saturated):
Lithium carbonate ---------------------- 1.54 g
Distilled water --------------------------- 100 ml
Mix well.
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 (Harris):
Ammonium or Potassium alum ------------ 100 g
Distilled water ------------------------------- 1000 ml
Heat to dissolve alum. Add 50 ml of 10% alcoholic hematoxylin solution and heat to boil for 1 minute. Remove from heat and slowly add 2.5 g of mercuric oxide (red). Heat to the solution and until it becomes dark purple color. Cool the solution in cold water bath and add 20 ml of glacial acetic acid (concentrated). Filter before use.
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 Harris hematoxylin solution for 8 minutes.
6. Wash in running tap water for 5 minutes.
7. Differentiate in 1% acid alcohol for 30 seconds.
8. Wash running tap water for 1 minute.
9. Bluing in 0.2% ammonia water or saturated lithium carbonate solution for 30 seconds to 1 minute.
10. Wash in running tap water for 5 minutes.
11. Rinse in 95% alcohol, 10 dips.
12. Counterstain in eosin-phloxine B solution (or eosin Y solution) for 30 seconds to 1 minute.
13. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each.
14. Clear in 2 changes of xylene, 5 minutes each.
15. Mount with xylene based mounting medium.
Results:
Nuclei ---------------------------------------- blue
Cytoplasm ---------------------------------- pink to red