Hematoxylin/Eosin staining experiment

Summary

This method of staining is based on the fact that tissue structures bind differently to different dyes. The dye hematoxylin stains basophilic structures blue-violet, while eosin stains acidophilic structures pink. Basophilic structures usually include parts containing nucleic acids, such as ribosomes, the nucleus, and ribonucleic acid (RNA)-rich regions of the cytoplasm.

Operation method

basic program

Materials and Instruments

Slides
Hematoxylin dye Ammonium hydroxide Eosin dye
Water bath Incubator

Move

1. Place the developed slide (in a slide holder) into a staining tray with water.

2. Prepare each of the following groups of liquids in the staining tray by number.

(1) Hematoxylin Staining Solution

(2) Water

(3) Water

(4) 0.1% NH4OH

(5) Water

(6) Water

(7) Eosin stain

(8) 95% ethanol

(9) 100% ethanol

(10) 100% ethanol

(11) Xylene

(12) Xylene

(13) Xylene3. Stain slides in hematoxylin staining solution for 20-30 s (do not stain too deeply), and immerse in water twice for 2 min each time.

4. Quickly immerse in 0.1% ammonium hydroxide and then in water.
5. Wash with water for 5 min, then stain with eosin for 20-30 s. '6. Dip the slide 8 times in the same dish of 95% ethanol, then 8 times in 100% ethanol, then 2 min in 100% ethanol for complete decolorization, and equilibrate in xylene 3 times for 2 min each.
Figure 1: Schematic diagram of slide fixation with fixation cutoff
7. Operate according to basic program 1, steps 5 to 9, for solid sheet processing.

Caveat

1. If immersed in 0.1% ammonium hydroxide for too long, the radioactive autoradiographic latex will detach from the slide.

2. If the staining is too dark, the slide can be immersed in 50% ethanol and decolorized for a few moments (observe the dye diffusion condition), if the staining is too weak, it can be re-stained.

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