Jul 02, 2025

Public workspaceHematoxylin and Eosin Staining (H&E) - Pregnant Female Reproductive TMC

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkrxx6v5r/v1
  • Donald Pizzo1,
  • Mana Parast1
  • 1University of California San Diego
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Cayla Mason
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkrxx6v5r/v1
Protocol Citation: Donald Pizzo, Mana Parast 2025. Hematoxylin and Eosin Staining (H&E) - Pregnant Female Reproductive TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkrxx6v5r/v1
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: June 11, 2025
Last Modified: July 02, 2025
Protocol Integer ID: 219973
Keywords: H&E, hematoxylin, eosin, pregnant female reproductive tmc, histological staining, protocol for hematoxylin, cytoplasmic cellular compartment, animal tissue technology center, other cellular structure, eosin staining, given tissue
Funders Acknowledgements:
Pregnant Female Reproductive Tissue Mapping Center
Grant ID: NIH U54 HD110347
Abstract
Here we describe the protocol for hematoxylin and eosin (H&E) histological staining employed by the UC San Diego Human & Animal Tissue Technology Center. H&E is used to distinguish nuclear and cytoplasmic cellular compartments and other cellular structures, as well as provide insights into overall patterns present within a given tissue.
Guidelines
Steps involving xylene or alcohol should be conducted in a fume hood; for the sake of simplicity, the author conducts all steps except water rinses in a fume hood.
Materials

AB
100% reagent alcoholFisher Chemical A962P-4
hematoxylinEpredia 7212
eosin Y with phloxineEpredia 71304
xyleneFisher Chemical X5-4
define solution (Clarifier 2)Epredia 7402
bluing solutionEpredia 7301
tap water
coverslip, 24 x 30mm #1.5VWR 48404-467
slidesFisher Scientific 12-550-15
coverslip mounting solutionCytoseal XYL 8312-4
Sakura 24 slide holderTissue-Tek 4465
slide staining dishesTissue-Tek 4456 or 4457


Troubleshooting
Safety warnings
  • XYLENE: Flammable, toxic, avoid contact with eyes and skin
  • 100% REAGENT ALCOHOL: Flammable, poisonous, avoid contact with eyes and skin
  • HEMATOXYLIN: Flammable, harmful vapors, poisonous, avoid contact with eyes, skin and clothing
  • EOSIN: Harmful vapor, avoid contact with eyes, skin, and clothing
  • BLUING REAGENT: Avoid contact with eyes and skin
  • DEFINE REAGENT: Avoid contact with eyes
Dry slides
8h
Place slides into a slide holder and air dry slides overnight prior to staining.
8h
Deparaffinization
31m
Prepare six slide staining dishes
  • Xylene (2)
  • 100% reagent alcohol (3)
  • Tap water (1)
5m
Submerge slides in the first xylene dish for 10 minutes. Gently mix up and down 3-5 times during incubation to facilitate wax removal.
10m
Remove slides and submerge in the second xylene dish for 5 minutes. Gently mix up and down 3-5 times during incubation to facilitate wax removal.
5m
Remove slides, draining off as much xylene as possible, and submerge in the first reagent alcohol dish for 2 minutes. Repeat twice more, removing and submerging into the next reagent alcohol dish, for a total of three submersions in reagent alcohol.
6m
Remove slides, submerge slides in water dish, remove slides, discard water, and refill dish with fresh tap water. Repeat 2 or 3 more times.
5m
Staining
1d 0h 59m
Prepare eleven staining dishes:
  • Hematoxylin (1)
  • Define solution (1)
  • Bluing solution (1)
  • Eosin-Y with phloxine (1)
  • 100% reagent alcohol (3)
  • Xylene (3)
  • Tap water (1)
Begin staining by submerging slides in hematoxylin dish for 1 minute.
1m
Remove slides from stain and rinse with running tap water until water runs clear, approximately 1 minute.
1m
Submerge slides in define solution for 1 minute.
1m
Remove slides, submerge slides in water dish, remove slides, discard water, and refill dish with fresh tap water. Repeat 2 or 3 more times.
5m
Submerge slides in bluing solution for 1 minute.
1m
Remove slides, submerge slides in water dish, remove slides, discard water, and refill dish with fresh tap water. Repeat 2 or 3 more times.
5m
Submerge slides in eosin for 1 minute.
1m
Remove slides, submerge slides in water dish, remove slides, discard water, and refill dish with fresh tap water. Repeat 2 or 3 more times.
5m
Air dry slides in slide holder for 15 minutes.
15m
Submerge slides in the first reagent alcohol dish for 1 minute. Repeat this step twice more, removing and submerging into the next reagent alcohol dish, for a total of three submersions in reagent alcohol.
3m
Air dry slides in slide holder for 15 minutes.
15m
Remove slides and submerge in the first xylene dish for 1 minute. Repeat this step twice more, removing and submerging into the next xylene dish, for a total of three submersions in xylene.
3m
Drain excess xylene and apply coverslip mounting medium.
3m
Apply coverslip to the slides and let air dry for 1 or 2 days.
1d