Mar 27, 2020

Public workspaceUF H&E Staining

DOI
dx.doi.org/10.17504/protocols.io.beamjac6
  • 1University of Florida
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Marda Jorgensen
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DOI: dx.doi.org/10.17504/protocols.io.beamjac6
Protocol Citation: Marda Jorgensen, Franchesca Farris 2020. UF H&E Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.beamjac6
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 26, 2020
Last Modified: October 02, 2020
Protocol Integer ID: 34861
Abstract

Hematoxylin and Eosin stains are the prefered method for histopathologic assessment of tissue sections.

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 identified 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
ReagentWater
ReagentXyleneFisher ScientificCatalog #X3P-1GAL
ReagentRichard-Allan Scientific™ High-Quality Dehydrants, Reagent AlcoholThermo FisherCatalog #9111
ReagentEosin-Y OPTIKCatalog #RS4359-A
ReagentHematoxylin OPTIK DKCatalog #RS4576-A
ReagentBluing Reagent OPTIKCatalog #RS-4363-B
ReagentAqueous ClarifierCatalog # RS4361-B
ReagentShandon MountantFisher ScientificCatalog #1900231
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).

- Xylenes and ethanol are flammable. Avoid open flames and perform procedure in a fume hood .
-Gloves should be worn when performing staining process
Before start
Confirm an adequate level of the reagent in each staining line station.

Verify that all slides are completely dry. Slides can be dried in an 55C oven for one hour prior to use.

Prepare staining line with reagents in order of use, based on steps three through 25.
Example of a H&E stain line.

Place slides into a xylene compatible slide rack.
Immerse slide holder in the first xylene staining station for 5 minutes.
5m
Immerse slide holder in the second xylene staining station for 5 minutes.
5m
Remove slide holder from xylene and transfer to the first staining station of 100% EtOH for 5 minutes.
Note
QUickly blot excess xylene from rack before transfering into ethanol.

5m
Transfer slide rack into the second EtOH station 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.


2m
Transfer to the second change of water for an additional 2 minutes.
Note
Quickly 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 de-ionized 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 station for 5 minutes.
Note
Be sure to blot excess ethanol before going into xylene.

5m
Transfer to xylene for 5 minutes.
5m
Transfer to a fresh xylene station for 5 minutes.
5m
After the second xylene change, use a xylene compatible mountant to affix a coverslip to the slide.