May 27, 2025

Public workspaceHematoxylin and eosin staining on FFPE sections

DOI
dx.doi.org/10.17504/protocols.io.q26g79jm3vwz/v1
  • 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
Weitao Lin
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.q26g79jm3vwz/v1
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
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

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 Duration02:00:00 at Temperature60 °C .
  • Ensure each jar has more than Amount80 mL of reagents for staining.

EasyDipâ„¢ slide staining jars.

Deparaffinization
Deparaffinization
37m
37m
Immerse slides in Xylene Dewax Jar 1 for Duration00:10:00 .

Note
Close Jar lid to minimise Xylene evaporation.

10m
Immerse slides in Xylene Dewax Jar 2 for Duration00:10:00 .

Note
Close Jar lid to minimise Xylene evaporation.

10m
Immerse slides in 100% Ethanol Dewax Jar 1 for Duration00:03:00 .
3m
Immerse slides in 100% Ethanol Dewax Jar 2 for Duration00:03:00 .
3m
Immerse slides in 96% Ethanol Dewax Jar 1 for Duration00:03:00 .
3m
Immerse slides in 96% Ethanol Dewax Jar 2 for Duration00:03:00 .
3m
Immerse slides in 70% Ethanol Dewax Jar for Duration00:03:00 .
3m
Immerse slides in H2O Jar for Duration00:02:00 .
2m
Staining and Dehydration
Staining and Dehydration
21m 50s
21m 50s
Immerse slides in Hematoxylin Jar for Duration00:01:00
1m
Rinse with deionized water in three H2O jars, keep the slides in the last jar with running deionized water for at least Duration00:05:00 in the sink. Check the hematoxylin staining under the microscope.
5m
Immerse in Bluing Buffer for Duration00:01:00 .

1m
Rinse with deionized water in three H2O jars, keep the slides in the last jar with running deionized water for at least Duration00:05:00 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 Duration00:00:30 .
30s
Immerse in Eosin Jar for Duration00:02:00 .
2m
Immerse in 96% EtOH Dehydration Jar 1 for Duration00:00:10 .
10s
Immerse in 96% EtOH Dehydration Jar 2 for Duration00:00:10 .
10s
Immerse in 100% EtOH Dehydration Jar 1 for Duration00:01:00 .
1m
Immerse in 100% EtOH Dehydration Jar 2 for Duration00:01:00 .
1m
Immerse in 100% EtOH Dehydration Jar 3 for Duration00:01:00 .
1m
Immerse in Xylene Dehydration Jar 1 for Duration00:02:00 .
2m
Immerse in Xylene Dehydration Jar 2 for Duration00:02:00 .
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
Storage and Imaging
1d
1d
Let slides dry for at least Duration24:00:00 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