Mar 09, 2026
Hematoxylin 26 Eosin Staining of Clinical Tissues in Formalin Fixed Paraffin Embedded (FFPE) Samples
- Ashleigh Abbott1,
- Robert Dalton1,
- Lauryn Lafayette1,
- Shane Priester1,
- Carlos Cruz2,3,
- Kiara Chan1,
- Kyle D. Allen1,2,4
- 1UF Department of Biomedical Engineering, College of Engineering;
- 2UF Pain Research and Intervention Center of Excellence;
- 3Department of Community Dentistry & Behavioral Science, College of Dentistry;
- 4Orthopedic and Sports Medicine Institute, College of Medicine
Protocol Citation: Ashleigh Abbott, Robert Dalton, Lauryn Lafayette, Shane Priester, Carlos Cruz, Kiara Chan, Kyle D. Allen 2026. Hematoxylin 26 Eosin Staining of Clinical Tissues in Formalin Fixed Paraffin Embedded (FFPE) Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l66m3zlqe/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: March 06, 2026
Last Modified: March 09, 2026
Protocol Integer ID: 270424
Keywords: Tissue staining, Knee Joint, Formalin fixed, Paraffin embedded, Hematoxylin, Eosin, Cartilage, Osteoarthritis, Formalin Fixed Paraffin Embedded, FFPE, cellularity, immune infiltration, vasculature, hematoxylin and eosin, H 26E, histological dyes hematoxylin, hematoxylin stain, cellular morphology in histological sample, hematoxylin, cytoplasmic components with high structural detail, identifying different cell, staining clinical ffpe sample, clinical tissues in formalin fixed paraffin embedded, extracellular matrix feature, cytoplasmic component, cellular morphology, extracellular matrix in orange, cytoplasm, different cell, evaluating tissue architecture, histological sample, surrounding extracellular matrix, samples this stain, tissue architecture, extracellular structure, stain, clinical tissue, eosin, staining, tissue type, collagen, connective tissue
Abstract
This stain is utilized when evaluating tissue architecture and cellular morphology in histological samples. Hematoxylin and Eosin (H 26E) staining helps not only in identifying different cell and tissue types, but also in visualizing nuclear and cytoplasmic components with high structural detail. This protocol describes the stepwise process for staining clinical FFPE samples to visualize cellular and extracellular structures using the histological dyes hematoxylin and eosin. Hematoxylin stains ribosomes and various other nuclear structures a deep blue-purple, while eosin stains the cytoplasm, collagen, connective tissue, and surrounding extracellular matrix in orange-pink to red hues. This method clearly demonstrates a broad range of cytoplasmic, nuclear, and extracellular matrix features, making H 26E great for pathologists.
Materials
- Slide holder cassettes
- Solution containers
- Slide coverslips
- Eyedropper
- Mounting medium (Permount)
- Fume hood
- Protocol 22 SafeClear Xylene Substitute (Fisher Healthcare, 68551-16-6, 11-002-220)
- Permount Mounting Medium (Fisher Chemical, 108-88-3, 68240-09-5, SP15-500)
- 5.00g Eosin Y
- 1.585L Ethanol
- 100mL Distilled Water
- 800mL Tap Water
- 300mL Xylene substitute
- 50mL Hematoxylin A
- 50mL Hematoxylin B
- 2.5mL Glacial acetic acid
- 1mL Hydrochloric acid
Troubleshooting
Safety warnings
NOTE: if staining is not going well (and you are following the protocol accurately) it is likely a processing (decal/infiltration/fixation) issue. Don 't make permanent changes to the protocol if this is the case.
Ethics statement
Tissue collection for this protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Before start
Note: solution prep is based on solution containers that fully submerge all sections on all slides being stained when filled with ~100mL. Ensure containers meet these standards or scale solution ratios to match these requirements.
Reagents
| Eosin Y disodium salt | VWR Life Science | 17372-87-1 | 0109-100G | ||
| Weigert’s Iron Hematoxylin A | Electron Microscopy Sciences | 64-17-5 67-56-1 517-28-2 | 26370-03 | ||
| Weigert’s Iron Hematoxylin B | Electron Microscopy Sciences | 7647-01-0 | 26458-05 | ||
| HistoPrep 100% ethanol | Decon Laboratories | 64-17-5 | UN1170 | ||
| Glacial acetic acid | Alfa Aesar | 64-19-7 | UN2789 | ||
| Hydrochloric acid | Fisher Chemical | A144S-500 | 7647-01-0 |
Solution prep
1h
Set aside from stock 200mL of 100% Ethanol
- Separate into 100mL aliquots
Using distilled water, mix from stock 300mL of 95% Ethanol (19:1)
- Separate into 100mL aliquots
Using distilled water, mix from stock 100mL of 90% Ethanol (9:1)
Using distilled water, mix from stock 300mL of 70% Ethanol (7:3)
- Separate into 100mL aliquots
Using distilled water, mix from stock 100mL of 50% Ethanol (1:1)
Combine 50 mL of Hematoxylin A and 50 mL of Hematoxylin B for 100mL of Weigert’s Hematoxylin (1:1)
- Wrap bottle in foil to prevent bleaching of dye after mixing
Combine 120mL of distilled water with 380mL ethanol
- On a stir plate with a magnetic stir bar, add 5g of Eosin Y to the previously prepared solution
- Allow powder to dissolve 10 minutes for your Eosin Y Stock Solution
In a separate container, combine 125mL of the Eosin Y Stock solution, 300mL of ethanol, 75mL of distilled water, and 2.5mL of Glacial Acetic Acid (50:120:30:1)
- This is your Eosin Y working solution
Mix 70mL of 100% Ethanol, 30mL of distilled water, and 1mL of Hydrochloric Acid for a ~1% Acid Alcohol solution (7:3:1)
Also Prepare:
- Five 100mL aliquots of Xylene substitute
- One 100mL aliquot of Distilled water
- Two 400mL aliquot of Tap Water
Set-up
30m
Have your chosen slides picked out and loaded into their slide holder
Set up your work station in the fume hood with solution containers lined up in order for the processing steps:
- Xylene substitute (3)
- 100% ethanol (1)
- 95% ethanol (1)
- 90% ethanol (1)
- 75% ethanol (1)
- 50% ethanol (1)
- Distilled water (1)
- Weigert’s hematoxylin (1)
- Large Tap Water Beaker (1)
- Acid Alcohol (1)
- Large Tap Water Beaker (1)
- Eosin Y Working Solution (1)
- Must be in a secondary containment vessel (due to glacial acetic acid and to contain the Eosin if you spill)
- 75% ethanol (2)
- 95% ethanol (2)
- 100% ethanol (1)
Label and fill solution containers with 100mL of their appropriate solutions, or specified volumes
Deparaffinize and hydrate slides
24m
To remove paraffin, soak slides in Xylene for 18 minutes, switching containers twice so 3 batches of Xylene each saturate the slides for 6 minutes each.
- Once the slides have sat in the first Xylene container for 6 minutes, remove and place them in the next container for 6 minutes
- Repeat once more.
18m
Next steps will gradually hydrate the slides, with carefully timed steps.
Leave slides in 100% ethanol for 1 minute, moving to the next container in line after the allotted time.
1m
Place slides in 95% ethanol for 1 minute, followed by 90%, 75%, and 50% ethanol, each for 1 minute.
4m
Now, move slides to the container of Distilled water for 1 more minute to complete the hydration.
1m
Staining tissue steps
10m 30s
Stain slides in Weigert’s hematoxylin for 5 minutes.
5m
Rinse slides in one of the Large water beakers for 60 seconds.
1m
Differentiate by placing slides in 1% Acid Alcohol for 1 minute.
1m
Then, rinse again in a large beaker of new tap water for 2.5 minutes.
2m 30s
Stain slides in Eosin Y working solution for 60 seconds.
1m
Dehydrate tissues
5m
Proceed to dehydrate in a similar fashion to the hydration steps, timing carefully
- 75% ethanol: 1 minute
- 75% ethanol: 1 minute
- 95% ethanol: 1 minute
- 95% ethanol: 1 minute
- 100% ethanol: 1 minute
5m
Lay slides flat to dry.
Cover slipping
After drying for hours, use and eyedropper and squeeze Permount along the length of the slide.
Carefully place a coverslip in one smooth motion to spread mounting medium,
Take care to avoid air bubbles, especially over the sample.
Place slides in cardboard slide storage holders to aid spread of Permount across coverslip.
Tip one of the edges of the holder up on its edge to further improve preservation.
Acknowledgements
National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant ID: UC2AR082196