Dec 13, 2025

Public workspacePost-PhenoCycler H&E

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld6oxog5b/v1
  • Joanna Bi1
  • 1Stanford University
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Joanna Bi
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld6oxog5b/v1
Protocol Citation: Joanna Bi 2025. Post-PhenoCycler H&E. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6oxog5b/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: December 13, 2025
Last Modified: December 13, 2025
Protocol Integer ID: 234911
Keywords: completed phenocycler assay, phenocycler assay, glass flow cell, assay, procedure
Abstract
This procedure is for removing the glass flow cells and performing H&E staining following the completed PhenoCycler assay.
Troubleshooting
Flow Cell Removal
Place sample slides in a staining dish or coplin jar containing Histoclear. Allow to sit overnight at room temperature.
Remove the glass flow cells from the sample slides. Use a razor blade if necessary
Rinse the sample slides in at least 1L of DI water
Wash the sample slides in 1x PhenoCycler Buffer without additive, 3 x 10 minutes.
Proceed to H&E immedietly or transfer slides to PhenoCycler Storage buffer and store 4ºC
H&E Staining
Prepare the following solutions in coplin jars, staining dishes, or an automatic slide stainer. Immerse the tissue completely and agitate occasionally. Depending on the manufacturer, adjust the incubation times for the hematoxylin and eosin as needed. Xylene steps must be performed in a fume hood.

Filter hematoxylin and eosin with filter paper before use. Change out hematoxylin and eosin per manufacturer's guidelines, or every 2 weeks. Change out all other solutions every usage. If staining a high volume of slides, change out other solutions multiple times as needed.
MilliQ water, 2 minutes
Mayer's hematoxylin, 15 minutes
MiiliQ water, 3 x 1 minute
Bluing solution, 1 minute
MilliQ water, 1 min
70% ethanol, 2 minutes
95% ethanol, 2 minutes
Eosin, 1 minute
95% ethanol, 2 x 30 seconds
100% ethanol, 2 x 30 seconds
Xylene, 2 x 3 minutes
Mount and coverslip. Allow to dry for at least 30 minutes room temperature.
Image in brightfield at 40X.