Hematoxylin stains negatively charged nucleic acids (nuclei & ribosomes) blue. Eosin stains proteins pink.

1. Paraffin blocks of tissue samples
2. Xylenes (Fisher Scientific X3S-4)
3. 200 Proof ethanol ( Fisher Scientific NC9255442 ). Also make 95% and 70% solution with dH₂O
4. Protocol® Hemotoxylin Harris ( Fisher Scientific 245-651)
5. Protocol® Eosin Y ( Fisher Scientific 245-658)
6. Clarifier 1 (Richard-Allan Scientific 7401)
7. Bluing Reagent (Richard-Allan Scientific 7301)
8. Permount ( Fisher Scientific SP15-100)
9. Microtome (Leica RM 2025)
10. Fisherbrand® Superfrost/Plus microscope slides, precleaned ( Fisher Scientific 12-550-15)
11. TissuePrep Flotation Bath 135 ( Fisher Scientific) (filled with MilliQ H₂O and heat to 42-43^oC)
12. Oven ( set to 60^o C)
13. Tissue-Tek II with pans and racks
14. Cover slips

1. Cut tissue blocks at the thickness of 5-7 microns and float on flotation bath.
2. Mark slides with pencil (xylenes can remove the ink marker).
3. Transfer sections onto slides and remove extra water with paper tower.
4. Bake slides in 60^oC oven for one hour then cool to room temperature.
5. Put slides in rack. Deparaffinize in xylenes, 2 changes for 10 min each.
6. 100% ethanol 10 min.
7. 95% ethanol 10 min.
8. 70% ethanol 10 min.
9. MilliQ H₂O 10 min.
10. Slightly overstain the sections with hematoxylin, usually 3-5 minutes.
11. Remove excess stain in running water, a beaker could be used.
12. Differentiate and destain a few seconds in clarifer 1 until sections look red, usually 4-5 dips.
13. Rinse 2 min in running water to remove the acid.
14. Blue in bluing reagent until nuclei stand out sharply blue (5 dips-2 min).
15. Wash in running water, 3 min.
16. Rinse in 95% ethanol 5-10 dips.
17. Stain in Eosin (2 dips to 2 min).
18. 95% ethanol 15-20 dips.
19. Absolute ethanol 15-20 dips x3.
20. Xylenes 10 dips x3.
21. Coverslip with Permount.
22. Sit overnight to dry.
    

Note and Readings:

A sharp blade is critical to cut sections. When the section's quality become bad, replace a new one to try. We use Accu-Edge® High Profile Microtome Blades from Sakura Finetek U.S.A., Inc, Torrance, CA 90501, Cat#4685

Virtual histology lab

The People of Hotel Intestine's protocol

MAYER'S HEMATOXYLIN AND EOSIN STAIN