Jan 14, 2025

Public workspaceYale University Hematoxylin and Eosin (H&E) Staining for Formalin-Fixed Paraffin-Embedded Human Lymph Node Tissues Protocol Adapted from VHIR-CIBERNED-ASAP

 Forked from Hematoxylin and Eosin (H&E) Staining
DOI
dx.doi.org/10.17504/protocols.io.5qpvo98x9v4o/v1
  • 1Yale University
  • Cellular Senescence Network (SenNet) Method Development Community
Negin Farzad
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo98x9v4o/v1
Protocol Citation: Negin Farzad, Archibald Enninful, Rong Fan 2025. Yale University Hematoxylin and Eosin (H&E) Staining for Formalin-Fixed Paraffin-Embedded Human Lymph Node Tissues Protocol Adapted from VHIR-CIBERNED-ASAP. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo98x9v4o/v1
Manuscript citation:
Bai Z, Zhang D, Gao Y, Tao B, Zhang D, Bao S, Enninful A, Wang Y, Li H, Su G, Tian X, Zhang N, Xiao Y, Liu Y, Gerstein M, Li M, Xing Y, Lu J, Xu ML, Fan R. Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues. Cell. 2024 Nov 14;187(23):6760-6779.e24. doi: 10.1016/j.cell.2024.09.001. Epub 2024 Sep 30. PMID: 39353436; PMCID: PMC11568911.
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: January 11, 2025
Last Modified: January 14, 2025
Protocol Integer ID: 118088
Funders Acknowledgements:
Yale TMC for Cellular Senescence in Lymphoid Organs
Grant ID: 1U54AG076043-01
Abstract
Hematoxylin and Eosin (H&E) Staining for for Formalin-Fixed Paraffin-Embedded Human Lymph Node Tissues after sectioning.
DEPARAFFINIZE
DEPARAFFINIZE
Bake the tissue section on the slide at 60 oC - 1 hr

Immerse the slide in Xylene_1 - 5 minutes
Immerse the slide in Xylene_2 - 6 quick immersions
HYDRATE
HYDRATE
Immerse the slide in 100% alcohol_1 - 6 quick immersions
Immerse the slide in 100% alcohol_2 - 6 quick immersions
Immerse the slide in 96% alcohol - 6 quick immersions
Immerse the slide in 70% alcohol - 6 quick immersions
Immerse the slide in 50% alcohol - 6 quick immersions
Immerse the slide in Distilled water (H₂O) - 2 minutes
STAINING
STAINING
Apply Harris Hematoxylin - according to preparation
Run the slide under tap water (H₂O) - 5 minutes
DIFFERENTIATE HEMATOXYLIN
DIFFERENTIATE HEMATOXYLIN
Apply Acid alcohol - 2 minutes. Acid alcohol is made by adding 0.3 ml of HCl to 250 ml of 70º alcohol.
Run the slide under tap water (H₂O) - 5 minutes
BLUEING
BLUEING
Apply NaHCO₃ - 1 minute. Add one tablespoon of NaHCO₃ to 250 ml of distilled water (H₂O).
Run the slide under tap water (H₂O) - 5 minutes
COUNTERSTAINING
COUNTERSTAINING
Immerse the slide in 96% alcohol - 3 minutes.
Apply Eosin - according to preparation
DEHYDRATE
DEHYDRATE
Immerse the slide in 96% alcohol - 6 quick immersions
Immerse the slide in 96% alcohol - 6 quick immersions
Immerse the slide in 100% alcohol - 6 quick immersions
Immerse the slide in 100% alcohol - 6 quick immersions
CLEAR
CLEAR
Immerse the slide in Xylene - until the preparations are clear. Leave in xylene until mounting.
Acknowledgements
The Hematoxylin and Eosin (H&E) Staining for Formalin-Fixed Paraffin-Embedded Human Lymph Node Tissues Protocol was done by Yale Pathology Tissue Services ( YPTS).