Dec 11, 2019
Version 2
UF / HuBMAP - H&E Staining Process V.2
- 1University of Florida
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Franchesca Farris, Marda Jorgensen, Leigh Propper 2019. UF / HuBMAP - H&E Staining Process . protocols.io https://dx.doi.org/10.17504/protocols.io.9qbh5sn
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 in our group and it is working.
Created: November 22, 2019
Last Modified: December 11, 2019
Protocol Integer ID: 30179
Keywords: h&e, hematoxylin, eosin, uf, ufl, hubmap, university of florida, npod
Abstract
This protocol will detail our method of utilizing the H&E stain as a nonspecific entity to achieve optimal results in our staining process for our research samples.
Hematoxylin and Eosin stains are used in many areas of the laboratory, including frozen sections, fine needle aspirates, and paraffin fixed embedded tissues. There are multiple factors involved in determining what makes a well stained slide, and one of the primary factors involved in the final product is pathologist preference. Other important factors also play important roles in your final stain, such as reagents and their quality, consistency, initial microtomy quality and selecting a sound procedure.
Hematoxylin is used to illustrate nuclear detail in cells. The resulting intensity and shade of the dye in the cells is primarily reliant on the length of time the sample spends in hematoxylin. The cytoplasmic component of the section is brought out by Eosin, which is the most commonly used counterstain in histology. It's varying shades of pink and red provide an exceptional contrast between the nuclear and cytoplasmic areas of interest.
Guidelines
-Managers and supervisors - are responsible for making sure that technicians are properly trained and equipment and facility are maintained in good working order.
-Laboratory personnel - are responsible for reading and understanding this SOP and related documents and to perform these tasks in accordance with the SOPs.
Materials
MATERIALS
Water
XyleneFisher ScientificCatalog #X3P-1GAL
Richard-Allan Scientific™ High-Quality Dehydrants, Reagent AlcoholThermo FisherCatalog #9111
Eosin-Y OPTIKCatalog #RS4359-A
Hematoxylin OPTIK DKCatalog #RS4576-A
Bluing Reagent OPTIKCatalog #RS-4363-B
Aqueous ClarifierCatalog # RS4361-B
Safety warnings
-Use universal safety precautions when handling human samples and personal protective equipment (e.g., face mask with shield, gloves, lab coat or apron).
-Use chemical and physical safety precautions when working with paraformaldehyde and sharps, respectively.
-Perform under a hood if available.
-Gloves should be worn when performing staining process
Before start
- Be sure to run a control slide prior to patient/research slides, or run at the same time.
- Confirm you have appropriate quantities of the reagents required.
-Verify that all slides have been dried in an oven prior to staining.
Ensure your staining line or staining set up has sufficient reagents and is correctly set up for use.
Example of a H&E stain line.
5m
Place slides into a xylene compatible slide rack.
30s
Immerse slide holder in the first xylene staining pot for 5 minutes.
5m
Immerse slide holder in the second xylene staining pot for 5 minutes.
5m
Remove slide holder from xylene and transfer to the first staining pot of 100% EtOH for 5 minutes.
Note
Be sure to blot excess xylene before going into ethanol.
5m
Remove slide rack from first EtOH dish, and transfer to the second EtOH pot for an additional five minutes.
5m
Transfer to 95% EtOH for 3 minutes.
3m
Transfer to 70% EtOH for 3 minutes.
3m
Transfer to 50% EtOH for 3 minutes.
3m
Transfer to water for 2 minutes.
Note
While sections are in water, skim surface of hematoxylin with a Kimwipe to remove oxidized particles.
2m
Transfer to the second change of water for an additional 2 minutes.
Note
Blot excess water from slide holder before going into hematoxylin.
2m
Transfer slides to Hematoxylin for 1 minute and 15 seconds.
1m 15s
Rinse in de-ionized water, and then transfer slides to tap water for 30 seconds.
30s
After rinsing in tap water, place slide rack back into the first dish of water for 1 minute.
1m
Transfer to second water staining dish for 1 minute.
1m
Transfer to Aqueous Clarifier for 45 seconds.
45s
Do a quick rinse in de-ionized water and then transfer to tap water for 1 minute.
1m
Transfer to bluing reagent for 1 minute.
1m
Place in water for 5 minutes.
5m
Transfer to 80% EtOH for 1 minute.
1m
Transfer to Eosin-Y for 1 minute.
1m
Transfer to 100% EtOH for 2 minutes.
2m
Transfer to the second 100% EtOH for 5 minutes.
Note
Be sure to blot excess ethanol before going into xylene.
5m
Transfer to xylene for 5 minutes.
5m
Transfer to next xylene pot for 5 minutes.
5m
After the second xylene change, use a xylene compatible mountant to affix a coverslip to the slide.
3m