Apr 21, 2025

Public workspaceMultiplexed Immunofluorescence Staining and Imaging of Fresh Frozen Tissue using Phenocycler Fusion 2.1.0 system  V.2

DOI
dx.doi.org/10.17504/protocols.io.x54v9rb24v3e/v2
  • 1Vanderbilt University
  • VU Biomolecular Multimodal Imaging Center / Spraggins Research Group
    Tech. support email: jeff.spraggins@vanderbilte.du
Thai Pham
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.x54v9rb24v3e/v2
Protocol Citation: Thai Pham, Anna Smith, Ellie Pringy, Jamie Allen, Angela R.S. Kruse, Melissa Farrow, Jeff Spraggins 2025. Multiplexed Immunofluorescence Staining and Imaging of Fresh Frozen Tissue using Phenocycler Fusion 2.1.0 system . protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9rb24v3e/v2Version created by Thai Pham
Manuscript citation:
PhenoCycler-Fusion User Guide (Version 2.1.0) 2023 by Akoya Biosciences, Inc.
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: February 17, 2025
Last Modified: April 21, 2025
Protocol Integer ID: 131047
Abstract
PhenoCycler User Guide for multiplex spatial proteomic detection on Fresh Frozen tissue sections
Pre-staining Procedure
Pre-staining Procedure
Download PhenoCycler-Fusion User Guide_2.1.0.pdfPhenoCycler-Fusion User Guide_2.1.0.pdf
Prepare Antibody Cocktail according to the corresponding section (page 35-36) of the PhenoCycler-Fusion User Guide (Version 2.1.0)
Prepare Buffer to pre-stain Fresh Frozen Tissue following the corresponding section (page 37) of the PhenoCycler-Fusion User Guide (Version 2.1.0)
Fresh Frozen Tissue Staining
Fresh Frozen Tissue Staining
Download PhenoCycler-Fusion User Guide_2.1.0.pdfPhenoCycler-Fusion User Guide_2.1.0.pdf
Follow the procedure In "Fresh Frozen Tissue Staining" section (page 39-44) of the PhenoCycler-Fusion User Guide (Version 2.1.0).
CODEX FF Tissue Imaging
CODEX FF Tissue Imaging
Download PhenoCycler-Fusion User Guide_2.1.0.pdfPhenoCycler-Fusion User Guide_2.1.0.pdf
Apply the flowcell onto tissue slide (page 29-39)
Instrument set-up for PhenoCycler scan (page 57-71)
Protocol references
PhenoCycler-Fusion User Guide (Version 2.1.0) 2023 by Akoya Biosciences, Inc.