Apr 16, 2024
Hematoxylin and Eosin (H&E) Staining of Tissues following Multiplexed Imaging on Phenocycler-Fusion
- 1Single Cell Biology Lab, The Jackson Laboratory, USA;
- 2The Jackson Laboratory
Protocol Citation: Emily Soja, Santhosh Sivajothi, William F Flynn, Elise T Courtois 2024. Hematoxylin and Eosin (H&E) Staining of Tissues following Multiplexed Imaging on Phenocycler-Fusion. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn55xgk5/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 09, 2024
Last Modified: April 16, 2024
Protocol Integer ID: 97990
Keywords: H&E, hematoxylin, eosin, codex, phenocycler, pohenocycler fusion, multiplex imaging
Abstract
Phenocycler-Fusion assay enables multiplexed antibody based imaging of FFPE or fresh frozen tissues. Hematoxylin and Eosin (H&E) staining is a gold standard method for observing tissue morphology. Obtaining multiplexed protein expression information and H&E images from the same tissue provides an additional layer of valuable information which can be used to interpret imaging data and used as input novel machine learning algorithms. This protocol details the steps to generate high quality H&E images from both FFPE and fresh frozen (FF) tissues following multiplexed imaging on the Phenocycler-Fusion.
This protocol can also be used for H&E staining following multiplexed imaging on other platforms. In that case, skip the 'Flow cell removal' section, perform two washes of 00:05:00 mins each in 1X PBS and proceed directly to Step 5.
Materials
Reagents:
- PBS (10X), pH 7.4 - Thermo cat# 70011044
- Xylene (Sigma cat# 534056) or Histo-Clear (National Diagnostics cat# HS-200)
- 100% (200 proof) ethanol - Sigma cat# E7023
- Hematoxylin Solution, Mayer′s - Sigma cat# MHS32-1L
- Epredia Shandon Bluing Reagent - Thermo cat# 6769001
- Eosin Y Solution; Alcoholic - Sigma cat# HT110116-500ML
- DPX Mountant - Sigma cat# 06522-100ML
- DI water
Materials and Equipment:
- Coplin jars
- Disposable transfer pipette- VWR cat# 10754-268
- Glass coverslip (1mm, 24 x 50) - Thermo cat# NC0106068
- Chemical fume hood
Flow cell removal
Flow cell removal
After a successful Phenocycler run, slides with attached flow cells can be stored in the storage buffer at 4 °C or used immediately for H&E staining.
The flow cell needs to be removed from the slide in order to continue with H&E staining. Retrieve tissue slides with attached flow cells either from the storage buffer or directly after Phenocycler run.
Fill a coplin jar with xylene or Histo-Clear. There must be sufficient volume to cover the entire flow cell.
Place slides in the coplin jar containing xylene or Histo-Clear and incubate for 24:00:00 hours at Room temperature
1d
Following incubation, gently remove the flow cell from the slide.
H & E staining
H & E staining
Place the slides in 100% ethanol for 00:02:00 mins . Raise and dip the slides 10 to 15 times to fully cover the tissue.
2m
Place the slides in 95% ethanol for 00:02:00 mins . Raise and dip the slides 10 to 15 times to fully cover the tissue.
2m
Place the slides in DI water for 00:02:00 mins . Raise and dip the slides 10 to 15 times to fully cover the tissue.
2m
Proceed immediately from step 5 and place the slides in the following reagents:
Mayer’s Hematoxylin - 00:04:00 mins
Note
Lymphoid tissues or tissues with dense cellularity should be stained for only 00:03:00 mins . Rinse the slide in DI water to observe the stain. Return the slide to Mayer's hematoxylin if staining is not adequate.
4m
DI water - 00:01:00 min
1m
Bluing Reagent - 00:01:00 min
1m
DI water - 00:01:00 min
1m
Alcoholic Eosin – 00:02:00 mins
2m
95% ethanol - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent
from previous step
1m
100% ethanol - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent from previous step
1m
100% ethanol - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent from previous step
1m
Xylene/Histo-Clear - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent from previous step
1m
Xylene/Histo-Clear - Hold in this solution as slides are mounted one by one
Slide mounting
Slide mounting
20m
To mount the slides, remove slides one at a time on to a paper towel inside the fume hood. Leave the other slides in the last xylene/Histo-Clear coplin jar to prevent over-drying the tissue.
Dry the back of the slide and tilt to remove excess xylene, but do not let the tissues dry.
Using a disposable transfer pipette add enough DPX mountant (or other xylene-based mountants) to cover the tissue area (1 to 2 drops)
Clean a glass coverslip quickly and remove any particles on its surface. Place the long edge of the coverslip on the edge of the slide closest to you, tip the slide towards yourself so the mountant begins to reach the coverslip, and gently push down until the mounting media has spread evenly across the slide area containing tissue.
Remove excess mountant by placing the edges along the paper towel. Avoid mountant covering the top of the coverslip.
Gently press down on the coverslip to remove any bubbles that remain above the stained tissue.
Allow mountant to cure inside the hood for at least 00:20:00 mins before moving and imaging. Xylene based mountants take up to 24 hours to cure completely.
20m
go to step #7 and repeat mounting procedure for remaining slides.
Mounted slides can be imaged and stored in a slide storage box at Room temperature
Expected Result:
H&E stained image of human pancreas FFPE tissue following multiplexed imaging on Phenocycler-Fusion (Data courtesy of Prof. Paul Robson, The Jackson Laboratory)
Overlay demonstrating multiplexed imaging and H&E staining on same tissue section (Data courtesy of Prof. Paul Robson, The Jackson Laboratory)