Staining Tissue With Hematoxylin and Eosin

Harris hematoxylin stains specimens rapidly with very good results. A commonly used stain is 4 mL of glacial acetic acid added to every 96 mL of Harris hematoxylin (see Note 4). In the progressive method, slides are first stained with hematoxylin, which stains only the nucleus, then rinsed and stained with eosin (21-23). The regressive method uses a stronger hematoxylin dye that stains the entire cell and an acid alcohol solution is used to remove the excess stain such that only the nucleus remains stained (21-23).

3.3.1. The Progressive Method

1. Dip in three changes of xylene for 2 min each.

2. 10 dips in absolute alcohol.

3. 10 dips each in two changes of 95% alcohol.

4. Rinse under tap water until water runs off evenly.

5. Dip for 1-3 min in Harris hematoxylin with acetic acid.

6. 10 dips each in two changes of tap water (see Note 5).

7. Dip in 0.25% ammonia water or 0.5% lithium carbonate until blue.

8. 10 dips each in two changes of tap water or running water may be used.

9. 10-20 dips in eosin or 1-3 min in eosin-phloxine.

10. 10-15 dips in two changes of 95% alcohol.

11. 10-15 dips each in three changes of absolute alcohol.

12. 10-15 dips each in three changes of xylene and let slides remain in the last container until a cover slip is applied.

13. The result from a representative section stained with hematoxylin and eosin is shown in Fig. 1A.

3.3.2. The Regressive Method

1. Dip in three changes xylene for 2 min each.

2. 10 dips in absolute alcohol.

3. 10 dips each in two changes of 95% alcohol.

4. Rinse under tap water until water runs off evenly.

5. 10-15 min in Harris hematoxylin without acetic acid.

6. 10 dips each in two changes of tap water.

7. 5-10 dips in 1% hydrochloric acid in 70% alcohol. 8 Wash well under running water.

9. Dip for 30 s in 0.25% ammonia water without agitating (see Note 6).

10. 10 dips each in two changes of tap water.

11. 10-20 dips in eosin.

A Hematoxylin and Eosin B Toluidinc blue

C Mast cell chymase

Leder stain fy * I 4

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Responses

  • Carl
    Why tissue dip into absolute alcohol,?
    7 years ago

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