May 27, 2025
Hematoxylin and eosin staining on FFPE sections
- Weitao Lin1,
- Yen Yeow2
- 1Systems Biology and Genomics Laboratory, Harry Perkins Institute of Medical Research, Nedlands WA 6009, Australia;
- 2Harry Perkins Institute of Medical Research
Protocol Citation: Weitao Lin, Yen Yeow 2025. Hematoxylin and eosin staining on FFPE sections. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g79jm3vwz/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: May 08, 2025
Last Modified: May 27, 2025
Protocol Integer ID: 217931
Keywords: Histology, H&E staining, stain of formalin, manual hematoxylin, staining process in 10x genomics visium hd workflow, hematoxylin, 10x genomics visium hd workflow, staining process, ffpe section, 10x genomic, ffpe sections this protocol, stain, eosin
Abstract
This protocol describes the steps for doing a manual Hematoxylin and Eosin (H&E) Stain of formalin-fixed paraffin-embedded (FFPE) tissue. This protocol is developed to utilise the reagents from the H&E staining process in 10x Genomics Visium HD workflow (CG000684).
Materials
Reagents and consumables
- Xylene, Reagent grade, Sigma 214736
- Ethanol, Pure (200 Proof, anhydrous), Sigma 534056
- Eosin Y solution, alcoholic, Sigma HT110116-500ML
- Gill II hematoxylin, Sigma GHS232
- Bluing buffer, Dako CS70230-2
- DPX mounting medium, Sigma 06522-100mL
- Coverslips
Equipment
- Chemical fume hood
- Microscope
- Easydipâ„¢ slide staining jars, Simpot M900-12
- Slide staining racks, M905-12DGY
Troubleshooting
Safety warnings
Appropriate precautions must be taken when working with xylene and ethanol. All staining procedures should be performed inside a certified fume hood to ensure proper ventilation. Nitrile gloves must be worn when handling xylene to prevent skin exposure.
Before start
- Ensure slides have been incubated in the oven for 02:00:00 at 60 °C .
- Ensure each jar has more than 80 mL of reagents for staining.
EasyDipâ„¢ slide staining jars.
Deparaffinization
37m
Immerse slides in Xylene Dewax Jar 1 for 00:10:00 .
Note
Close Jar lid to minimise Xylene evaporation.
10m
Immerse slides in Xylene Dewax Jar 2 for 00:10:00 .
Note
Close Jar lid to minimise Xylene evaporation.
10m
Immerse slides in 100% Ethanol Dewax Jar 1 for 00:03:00 .
3m
Immerse slides in 100% Ethanol Dewax Jar 2 for 00:03:00 .
3m
Immerse slides in 96% Ethanol Dewax Jar 1 for 00:03:00 .
3m
Immerse slides in 96% Ethanol Dewax Jar 2 for 00:03:00 .
3m
Immerse slides in 70% Ethanol Dewax Jar for 00:03:00 .
3m
Immerse slides in H2O Jar for 00:02:00 .
2m
Staining and Dehydration
21m 50s
Immerse slides in Hematoxylin Jar for 00:01:00
1m
Rinse with deionized water in three H2O jars, keep the slides in the last jar with running deionized water for at least 00:05:00 in the sink. Check the hematoxylin staining under the microscope.
5m
Immerse in Bluing Buffer for 00:01:00 .
1m
Rinse with deionized water in three H2O jars, keep the slides in the last jar with running deionized water for at least 00:05:00 in the sink.
Note
Carryover of the bluing reagent can raise the pH of eosin, resulting in a weak eosin staining.
5m
Dip in 96% EtOH Before Eosin Jar for 00:00:30 .
30s
Immerse in Eosin Jar for 00:02:00 .
2m
Immerse in 96% EtOH Dehydration Jar 1 for 00:00:10 .
10s
Immerse in 96% EtOH Dehydration Jar 2 for 00:00:10 .
10s
Immerse in 100% EtOH Dehydration Jar 1 for 00:01:00 .
1m
Immerse in 100% EtOH Dehydration Jar 2 for 00:01:00 .
1m
Immerse in 100% EtOH Dehydration Jar 3 for 00:01:00 .
1m
Immerse in Xylene Dehydration Jar 1 for 00:02:00 .
2m
Immerse in Xylene Dehydration Jar 2 for 00:02:00 .
2m
Keep the slides in Xylene Clearing Jar. Apply DPX mounting medium onto the section, and place the coverslip onto the mounting medium slowly to avoid generating any bubble with tweezers.
Note
Remove any dust on the coverslip with an air blower.
Storage and Imaging
1d
Let slides dry for at least 24:00:00 for the mounting medium to cure before imaging on the slide scanner. Before scanning slides, ensure no DPX mounting medium is overhanging the slide. Scrape off excess mounting medium with a blade.
1d