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