Hematoxylin and Eosin (H&E) staining of Frozen sections:

Why do an H&E?

Well, don't you want to identify the nature of the tissue section first, so that you can then proceed with decisions on further stains and / or immunostains. The H (in the meatoxylin) stains all nuclei within the tissue, and then the E (in the Eosin) stains the cytoplasm, and results in good contrast.

NOTE: An H&E stain, in fact, any dye used to stain tissue sections, will generate fluorescent signals. Thus dye stained sections are great to have on hand when trying to focus on specimens that have been labeled for viewing using epifluorescence light

After completing an IMMUNOSTAIN on tissue sections, NO EOSIN is used, since it will confound the results.

After completing an immunostains on tissue sections, and if the colored SUBSTRATE used is AQUEOUS, remember that the Hematoxylin that is used to stain the nuclei in immunostained tissue section is also AQUEOUS (Mayers NOT Gill's).

1. Fix frozen sections in 10% buffered formalin (Fisher SF93-4) for 20 minutes and wash in water
2. Stain nuclei by immersing in GILL II HEMATOXYLIN (Surgipath 01522) for 3 minutes.
3. wash in water and immerse in blueing agent (Fisher CS410-4) for 30 seconds, and wash in water
4. Immerse in 95% ethanol briefly
5. Stain cytoplasm by immersing in EOSIN (Surgipath 01602) for 3 minutes and dip once quickly in water
6. dehydrate by immersing in 1 changes of 95% ethanol for 1 min, and then in 3 changes of 100% ethanol (Fisher A962p) for 10 dips each.
7. Immerse in 3 changes of Citrosol (Fisher 22-143-975) for 2 minutes each
8. Mount slides with Cytoseal 60 (VWR 48212-154) using glass coverslips (Surgipath 00145)

Hematoxylin / Eosin staining of PARAFFIN sections:

1. Deparaffinize by immersing successively, in 2 changes of xylene (Fisher X3P) for 10 minutes each
2. Rehydrate by immersing in decreasing concentrations of ethanol (Fisher A962p) :
3. 100% for 5 minutes 100% for 5 minutes 95% for 5 minutes 95% for 5 minutes
4. Immerse in water for 5 minutes
5. Follow steps 2 through 8 above.

Hematoxylin and eosin stain on frozen sections for Laser Capture microscopy

(American Journal of Pathology February 2000 page 446):

1. Thaw frozen sections ( on plain untreated glass slides) one at a time to decrease degradation
2. Fix with 70% ethanol for 10 seconds
3. wash in deionized water
4. immerse in fresh MAYERS hematoxylin for 30 seconds
5. wash in water
6. Immerse in blueing solution for 30 seconds
7. wash in 70% ethanol
8. immerse in eosin for 90 seconds
9. dehydrate sections with two 10 second washes in 95% ethanol and two 10 second washes with 100% ethanol
10. place in xylene for 30 seconds

A GOOD REFERENCE FOR OTHER HISTOCHEMICAL STAINS:

https://www.leicabiosystems.com/knowledge-pathway/special-stains-which-one-how-and-why-part-ii-connective-tissue/