Oct 04, 2023

Public workspaceBuck Institute Morphology H & E staining protocol V.3

DOI
dx.doi.org/10.17504/protocols.io.36wgq3n2ylk5/v3
  • 1Buck Institute for Research on Aging
  • Stella Breslin: Morphology Core standard protocol
Stella Breslin
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DOI: dx.doi.org/10.17504/protocols.io.36wgq3n2ylk5/v3
Protocol Citation: Stella Breslin, Akos A Gerencser 2023. Buck Institute Morphology H & E staining protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3n2ylk5/v3Version created by Akos A Gerencser
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: September 28, 2023
Last Modified: October 04, 2023
Protocol Integer ID: 88551
Keywords: FFPE, hematoxylin, eosin, H&E, stain, section, histology, slide
Abstract
Histological Stain for FFPE slides
RESULTS: Nuclei-blue; Cytoplasm, red blood cells and connective tissue-shades of pink
Materials
1. Modified Mayes’s Hematoxylin HXMMHPT StatLab Medical Products
2. 10% Acidic Acid
3. Scott’s Tap Water (1L + 3.5g Sodium Bicarb + 20g Magnesium Sulfate pH 8.0) - Bluing Agent
4. Eosin Y Stain STE0150 StatLab Medical Products
5. reagents in 250 ml Coplin Jar
Safety warnings
Xylene (C8H10)CAS No. 1330-20-7 is a colorless, flammable liquid with a sweet odor. Exposure to xylene can irritate the eyes, nose, skin, and throat. Xylene can also cause headaches, dizziness, confusion, loss of muscle coordination, and in high doses, death. Workers may be harmed from exposure to xylene.
from https://www.cdc.gov/niosh/topics/xylene/
Permount Mounting Media is toluene-based synthetic resin mounting medium. Use caution around uncured media.
https://www.cdc.gov/niosh/topics/toluene/
Before start
Slides can be placed in 60 C oven prior to deparaffinization.
Hematoxylin and Eosin Staining
Hematoxylin and Eosin Staining
Deparaffinization and rehydration:
  1. 2 x xylene 7 min,
  2. 100% ethanol 4 min,
  3. 95% 4 min,
  4. 80% 4 min,
  5. 70% 4 min,
  6. bring to water.
Hematoxylin staining for 30 seconds - 4 min. using filtered dye (usually 2 mins) - Modified Mayes’s Hematoxylin HXMMHPT StatLab Medical Products
Rinse in ddH20 x2
10% acetic acid rinse (acid into water) – 1 min
rinse in ddH20 x2
TBS (1x) rinse or Scott’s Tap Water (1L + 3.5g Sodium Bicarb + 20g Magnesium Sulfate pH 8.0) - 2 mins
Rinse in ddH20
Rinse in 70% ETOH - 1 min
Stain Eosin for 0.5 to 2 minutes (keep an eye on intensity of pink) – Eosin Y Stain STE0150 StatLab Medical Products
Rinse in 90% EtOH
Rinse in
  1. 2 x 95% EtOH,
  2. 2 x 100% EtOH,
  3. xylene
Coverslip with Permount Mounting Medium EMS #17986-01