Protocol Citation: Sarah Johnston, Alexander Cabrera, Kate O'Neill 2025. Post-PhenoCycler Fusion Manual Hematoxylin and Eosin (H&E) Staining of Female Reproductive System Tissues . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzqrd2vx1/v1
Manuscript citation:
Citation
Andrew Houston. Akoya Biosciences PhenoCycler Fusion (formerly CODEX) User Guide. protocols.io.
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: May 13, 2025
Last Modified: May 15, 2025
Protocol Integer ID: 218198
Keywords: H&E, CODEX, Akoya, Histology, tissue sections with hematoxylin, histopathological inspection of tissue morphology, staining formalin, female reproductive system tissue, manual hematoxylin, hematoxylin, histopathological inspection, tissue morphology, akoya phenocycler, tissue section, removal of the akoya flowcell, fusion imaging, akoya flowcell, eosin, slide staining, tissue
Funders Acknowledgements:
Human BioMolecular Atlas Program (HuBMAP) Method Development Community Human BioMolecular Atlas Program (HuBMAP)
Grant ID: U54HD104392
Abstract
This protocol describes a standardized method for staining formalin-fixed paraffin-embedded (FFPE) tissue sections with hematoxylin and eosin (H&E) after Akoya Phenocycler-Fusion imaging. The procedure allows for removal of the Akoya flowcell and does not require it to remain mounted on the slide. H&E staining following multiplex imaging enables histopathological inspection of tissue morphology and integrity.
Guidelines
Use appropriate handling and personal protective equipment when handling any human samples.
Use appropriate handling and personal protective equipment when any flammable substance :Xylenes (ACS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #247642-2.5LEthanol Decon LabsCatalog #2716
Make sure you fully complete the Akoya's PhenoCycler Fusion's protocol and imaging prior to separating the flowcell. H&E staining of the tissue will render the sample unsuitable for processing PhenoCycler technology.
Research involving humans and animals must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
The sample collection for The Human Biomolecular Atlas Program (HuBMAP): Multi-scale Molecular Mapping of the Female Reproductive System were approved by the University of Pennsylvania Institutional Review Board IRB00000039 (IRB Protocol #844297), and written informed consent was obtained prior to participation.
Removing Phenocycler-Fusion flowcell
12h
Post-imaging in the Phenocycler Fusion system, store slide Overnight in Xylenes (ACS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #247642-2.5L at Room temperature to dissolve flowcell adhesion strip.
12h
H&E Staining
37m 30s
Rehydration
Soak in Xylenes (ACS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #247642-2.5L for 00:05:00 in a slide holder at Room temperature , repeat soak for a total of two soaks.
10m
Soak in 100% Ethanol for 00:02:00 in a slide holder at Room temperature, repeat twice for a soak for a total of three times.
6m
Soak in 95% Ethanol for 00:02:00 in a slide holder at Room temperature.
2m
Soak in 80% Ethanol for 00:02:00 in a slide holder at Room temperature.
2m
Soak in 70% Ethanol for 00:02:00 in a slide holder at Room temperature.
2m
Soak in deionized water (dH20) in a slide holder twice at Room temperature.
1m
Staining
Place slide on stain box, pipette between 200 µL to 400 µL of Mayer’s HematoxylinDakoCatalog #S3309 on top of the slide and incubate for 00:02:30 at Room temperature (this step may be optimized for 1 to 5 minutes)
3m
Wash excess Hematoxylin by dipping the slide in dH20 in a slide holder a total of three times at Room temperature. Use fresh dH20 for each dip.
1m 30s
12 dips in Acid alcohol at Room temperature.
30s
Rinse under Room temperature tap water for 00:02:00 and 3 dips in dH20
2m 30s
Place back slide on stain box, pipette between 200 µL to 400 µL of Eosin Y solution aqueousMerckCatalog #HT110216-500ML and incubate for 00:01:00 at Room temperature.
1m
Dip once in dH20 at Room temperature to wash off excess Eosin Yand immediately proceed to dehydration.
Dehydration and mounting
Soak in 95% Ethanol for 00:02:00 in a slide holder at Room temperature.
2m
Soak in 100% Ethanol for 00:02:00 in a slide holder at Room temperature.
2m
Soak in Xylenes (ACS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #247642-2.5L for 00:02:00 in a slide holder at Room temperature.
2m
Air dry completely and mount with 60 µL to 100 µL of Permount mounting mediumFisher Scientificand place the coverslip by angling the coverslip and let fall gently onto the slide. Allow the Permount to spread beneath the coverslip, covering all the tissue. Seal slide with clear nail polish and image
Protocol references
Citation
Sarah Black, Darci Phillips, John W. Hickey, Julia Kennedy-Darling, Vishal G. Venkataraaman, Nikolay Samusik, Yury Goltsev, Christian M. Schürch, Garry P. Nolan (2021). CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nature Protocols.
Sarah Black, Darci Phillips, John W. Hickey, Julia Kennedy-Darling, Vishal G. Venkataraaman, Nikolay Samusik, Yury Goltsev, Christian M. Schürch, Garry P. Nolan. CODEX multiplexed tissue imaging with DNA-conjugated antibodies
Fang Ping from the Wistar Institute for slicing and mounting of FFPE Female Reproductive System sections.
This research was supported by the National Institutes of Health (NIH) common fund through the Office of Strategic Coordination, Office of the NIH Director under award U54HD104392.
