Apr 18, 2024
OSU TriState SenNet H&E staining of Formalin-Fixed, Paraffin-Embedded (FFPE) tissue sections
- Lorena Rosas1,
- Ana L. Mora1,
- mauricio.rojas1
- 1Division of Pulmonary, Critical Care and Sleep Medicine, College of Medicine, Ohio State University, Columbus, Ohio, USA.
- TriState SenNet
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Lorena Rosas, Ana L. Mora, mauricio.rojas 2024. OSU TriState SenNet H&E staining of Formalin-Fixed, Paraffin-Embedded (FFPE) tissue sections. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzpjngpk/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: April 18, 2024
Last Modified: April 18, 2024
Protocol Integer ID: 98457
Keywords: H&E, FFPE, TriState, SenNEt, tissue sections, staining
Funders Acknowledgements:
TriState SenNET (Lung and Heart) Tissue Map and Atlas consortium - NIH
Grant ID: Grant ID: U54AG075931
Abstract
Hematoxylin and eosin (H&E) stains are essential for recognizing the different tissue types and morphologic changes that contribute to the diagnosis of many diseases. The presence of two-component dye gives the advantage of differentially stains tissue components. Hematoxylin stains nucleic acid and ribosomes in blue, while eosin is stains proteins, such as
collagen and elastin in pink. This protocol describes manual H&E staining of tissue sections from Formalin-Fixed, Paraffin-Embedded (FFPE) block.
Materials
XyleneFisher ScientificCatalog #X5P-1GAL
100% Ethanol Fisher ScientificCatalog #22-032-601
Hematoxylin solution according to MayerMerck MilliporeSigma (Sigma-Aldrich)Catalog #51275-500ML
Acetic Acid 100%Merck MilliporeSigma (Sigma-Aldrich)Catalog #A6283
Epredia™ Shandon™ Bluing ReagentFisher ScientificCatalog #6769001
Eosin Y solution aqueousMerckCatalog #HT110216-500ML
Chemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
Fisherbrand™ Superslip™ Cover SlipsFisher ScientificCatalog #12-541-055
KimwipesKimberly-ClarkCatalog #34120
Equipment
BZ-X800
NAME
Microscope
TYPE
Keyence
BRAND
BZ-X800
SKU
Safety warnings
- Use universal safety precautions when handling tissue specimens and personal protective equipment (PPE).
- All steps with flammable reagents, such as xylene/ethanol, must be done inside a fume hood.
Staining Procedure
Staining Procedure
Prepare slides
Place the slides at 60 °C for 30 minutes.
The solutions are fill in square glass staining jars.
Place slides in glass staining racks.
Deparaffinization of tissue slides: Remove remaining paraffin wax from tissues.
Place slides into a Xylene for 10 min, three times.
Remove Xylene and replace from ethanol (EtOH)
Place slides to 100% EtOH for 5 min, two times.
Rehydration of tissue slides: Remove alcohol from tissue and replace alcohol from water.
Place slides to 95% EtOH for 3 min, two times.
Place slides to 70% EtOH for 3 min, two times.
Place slides to Distilled H2O (DI) for 1 min, two times.
Nuclear Stain: Hematoxylin is a basic dye, so it can bind and stain acid components of the cell such as nucleic acid and ribosomes.
Place slides to Mayer’s Hematoxylin for 8 min.
Place slides to DI H2O for 30 sec, two times.
Then rinse in tap water for 5 min.
Differentiation
Place slides to 5% Acetic Acid, 3 quick dips.
Place slides to DI H2O for 30 sec, two times.
Then rinse in tap water for 3 min.
Bluing: Step important to give, cool bluish purple color to the acidic cellular components.
Place slides to Bluing Reagent for 2 min.
Then rinse in tap water for 3 min.
Counting staining: Is to give a pink color and this counterstain also helps to differentiate between nuclei and non-nuclear components in cells.
Place slides to Eosin Y solution aqueous for 2 min.
Place slides to DI H2O for 30 sec, two times.
Differentiation of eosin: Removes unnecessary eosin – check under microscope.
Place slides to 95% EtOH for 3 min, two times.
Dehydration of tissue slides: Remove water with ascending concentration alcohol.
Place slides to 70% EtOH for 2 min, two times.
Place slides to 95% EtOH for 2 min, two times.
Place slides to 100% EtOH for 2 min, two times.
Clearing: To have clear background and to remove alcohol from tissue sections.
Place slides to Xylene for 2 min, two times.
Use Kimwipe to absorb some excess of Xylene. “Do not allow the tissue section to dry out”.
Add ~30uL of mounting medium on the tissue.
Place the coverslip, the medium will spread gradually to the edges of the coverslip. “Avoid air bubbles underneath the cover slip”.
Imaging
Imaging
Slides are scanned using a Keyence BZ-X800 microscope. Images are acquired with color brightfield using a 10X objective.