H&E Staining Method and Protocol - Harris

 

NovaUltra Special Stain Kits

 

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):

      Potassium or ammonium (alum) ----------- 100 g

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

      Heat to dissolve. 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

 

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