Hematoxylin and Eosin Stain
Use as a differential stain or a counterstain on paraffin sections or mounted vibratome sections.
Delafield's Hematoxylin.
This may be used for nuclear counterstain, Nissl stain or in conjunction with eosin for histologic morphology.
(from Lillie, P. 206, Table 7-6)
Hematoxylin
Hematoxylin 6.4 gm
Ammonium alum (NH4)2SO4.Al2(SO4)3.24(H2O) 60 gm
Ethanol 200 ml
Glycerol 160 ml
Distilled water 640 ml
Mix for quite awhile (about 2-4 hrs). Allow the stain to ripen in the dark for 6-8 weeks before using. Although it will work immediately after mixing the resulting stain is somewhat dull.
Acid Alcohol
Conc. HCl 4 ml
95% Ethanol 396 ml
Blueing Agent
Sodium bicarbonate 1 gm
distilled water 1 liter
Eosin Y stock (from Humason, P. 141)
Eosin Y, C.I. 45380 1 gm
70% Ethanol 1 liter
Glacial acetic acid 5 ml
Eosin Y working solution:
Dilute Eosin stock solution 1:1 with 70% ethanol, then add 2-3 drops of glacial acetic acid.
Staining.
1. Slides are deparaffinized and rehydrated, frozens or vibratome sections are best mounted on slides and rehydrated;
DO NOT USE POLY-L-LYSINE SUBBED SLIDES FOR EOSIN STAINING. THE PLL PICKS UP EOSIN AND WILL CREATE A PINK TO ORANGE BACKGROUND BEHIND THE TISSUE SECTIONS!
2. Slightly overstain the sections with hematoxylin, usually 3-5 minutes, depending upon section thickness and fixative (up to 20 min. if solution is not fully ripened);
3. Remove excess stain in tap water, 2 min.;
4. Differentiate and destain a few seconds in acidic alcohol until sections look red, usually 4-5 dips;
5. Rinse briefly in tap water to remove the acid;
6. Blue in bicarbonate until nuclei stand out sharply blue, about 2 min.;
7. Rinse in running tap water, 8 min.;
8. Dehydrate and clear, or stain with eosin.
Eosin staining:
SEE NOTE ABOVE REGARDING USE OF PLL SUBBED SLIDES.
1. Take hematoxylin stained slides from the last tap water rinse and place in 70% ethanol for 3 min.;
2. Place slides in eosin for 2 min.;
3. Take slides through 3 changes of 95% ethanol, 5 min. each;
4. Then transfer to the first absolute ethanol of the clearing
series.
Testing hematoxylin stain solution to see if it is still usable.
Add several drops of stain to tap water (not distilled or deionized). If they turn bluish-purple immediately, it is satisfactory. However, if they change slowly, stays reddish or brownish, then the stain should be discarded.