Nov 27, 2025
UMN TMCs - Histology H&E Staining Protocol
- BLS Histology1,2
- 1Clinical and Translational Science Institute;
- 2University of Minnesota, Minneapolis, MN
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: BLS Histology 2025. UMN TMCs - Histology H&E Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyqmwg5r/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: November 19, 2025
Last Modified: November 27, 2025
Protocol Integer ID: 232973
Keywords: staining protocol, technique in histology, staining process, staining technique, histology, procedure for hematoxylin, staining differentiate, staining, hematoxylin, visualizing tissue structure, differentiates between cellular nuclei, tissue structure, eosin, umn tmc, cytoplasm, connective tissue, lab safety
Abstract
This SOP describes the procedure for Hematoxylin and Eosin (H &E) staining, a widely used technique in histology for visualizing tissue structure. H &E staining differentiates between cellular nuclei (stained blue) and cytoplasm/connective tissue (stained pink to red). The SOP ensures standardization of the H &E staining process to produce high-quality, consistent results. Training on lab safety, chemical handling, and staining techniques is required for implementation.
Guidelines
Training:
1. Personnel must complete training on lab safety protocols, including the handling of hazardous chemicals under a fume hood and the use of PPE.
2. Trainees will observe a trained technician, practice under supervision, and pass a practical test to demonstrate proficiency in the H &E staining protocol.
This protocol may require prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Chemicals and Solutions:
- Harris Alum Hematoxylin
- Acid Alcohol (70% alcohol + concentrated hydrochloric acid)
- Ammonium Hydroxide (0.2% ammonia water)
- Alcoholic Eosin
- Xylene
- Ethanol (100%, 95%, 80%)
- Glacial Acetic Acid
Equipment:
- 60°C oven
- Fume hood
- Personal protective equipment (PPE): goggles, gloves, mask
Troubleshooting
Procedure
Preparation of Slides:
Place slides in a 60°C oven to dry for 20 minutes.
Deparaffinization and Hydration:
Immerse slides in three changes of xylene, 5 minutes each.
Progress through two changes of 100% ethanol (3 minutes each), followed by 95% ethanol (3 minutes), and 80% ethanol (5 minutes).
Rinse slides in tap water for 5 minutes.
Hematoxylin Staining:
Stain slides with Harris hematoxylin solution for 5 minutes.
Rinse in running tap water.
Differentiation:
Dip slides in 1% acid alcohol for two quick dips to decolorize.
Bluing:
Dip slides in 0.2% ammonia water twice.
Rinse in running tap water for 5 minutes.
Dehydration and Counterstaining:
Place slides in 80% ethanol for 1 minute.
Counterstain with eosin Y working solution for 1 minute.
Final Dehydration and Clearing:
Dehydrate through one change of 80% ethanol (5 dips), two changes of 95% ethanol (8-10 dips each), and two changes of 100% ethanol (10-15 dips each).
Clear slides in two changes of xylene (15 dips each), then transfer to a third xylene bath for coverslipping.
Mounting:
Mount coverslips with a xylene-based medium.