Apr 15, 2022
Version 2
H&E Staining for Pancreas or Eye Cryosections V.2
- Diane Saunders1,
- Angela R.S. Kruse1,
- Jamie Allen1,
- Carrie Romer1,
- Danielle Gutierrez1,
- Alvin Powers1,
- Jeff Spraggins1
- 1Vanderbilt University
Protocol Citation: Diane Saunders, Angela R.S. Kruse, Jamie Allen, Carrie Romer, Danielle Gutierrez, Alvin Powers, Jeff Spraggins 2022. H&E Staining for Pancreas or Eye Cryosections. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz3e6rgx1/v2Version created by Carrie Romer
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 15, 2022
Last Modified: October 18, 2023
Protocol Integer ID: 60846
Keywords: HuBMAP, BIOMIC, Vanderbilt, Pancreas, H&E staining, Eye, Retina
Abstract
This protocol for H&E staining can be applied to either fixed or unfixed frozen cryosections.
Materials
1. Coplin jars
2. 95% Ethanol
3. Hematoxylin
4. Bluing Solution
5. Eosin
6. 100% Ethanol
7. Xylene
8. Coverslips
9. Cytoseal
Safety warnings
1.Safety glasses or goggles, proper gloves, and a lab coat required. The area should be adequately vented and a lab mat placed underneath all solutions.
2. Xylenes should be used in the fume hood.
Air dry sections for 00:05:00 .
5m
Incubate in 95% alcohol for 00:01:00 , then wash, dipping until clear.
1m
Incubate in hematoxylin for 00:00:30 , then wash until clear.
30s
Dip 3-4x in bluing solution, then wash for 00:00:30
30s
Dip 3-4x in 95% alcohol.
Dip 1-2x in eosin.
Dip 5-10x in 95% alcohol, then repeat using 2nd aliquot.
Dip 5-10x in 100% alcohol, then repeat using 2nd aliquot.
Dip 5-10x in xylene, then repeat using 2nd aliquot.
Apply coverslip and seal.
Figure 1. H&E stained pancreas with magnified section showing nuclei in blue and cytoplasm in pink.
Figure 2. H&E stain of human retina tissue from a 72 year old donor. Dark purple nuclear staining and pink cytoplasmic staining allow the user to readily distinguish retinal layers such as (from bottom to top) the: sclera, choroid, retinal pigmented epithelium, photoreceptor layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, and nerve fiber layer.