May 24, 2022

Public workspaceCODEX® Multiplexed Imaging | Modality Overview V.1

DOI
dx.doi.org/10.17504/protocols.io.36wgq7dryvk5/v1
  • Diane Saunders1,
  • Conrad Reihsmann2,
  • Alexander Hopkirk2,
  • Marcela Brissova1,
  • Alvin C. Powers1
  • 1Vanderbilt University, Vanderbilt University Medical Center;
  • 2Vanderbilt University Medical Center
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DOI: dx.doi.org/10.17504/protocols.io.36wgq7dryvk5/v1
Protocol Citation: Diane Saunders, Conrad Reihsmann, Alexander Hopkirk, Marcela Brissova, Alvin C. Powers 2022. CODEX® Multiplexed Imaging | Modality Overview. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7dryvk5/v1
Manuscript citation:
Walker JT, Saunders DC, Rai V et al. RFX6-mediated dysregulation defines human β cell dysfunction in early type 2 diabetes. Biorxiv (2021) doi:10.1101/2021.12.16.466282.
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: March 25, 2022
Last Modified: May 24, 2022
Protocol Integer ID: 59918
Abstract
This is an overview of all protocols currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group for performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). It includes links to each of the individual protocols that make up the workflow for various applications and projects.
Procure and/or prepare samples fixed with 4% paraformaldehyde (PFA).

HuBMAP | VU TMC Eye/Pancreas | Human Pancreas Processing for Multiple Applications (HuBMAP)
Mouse Pancreas Dissection and Fixation for Cryosectioning (IPA Core)

Note
4% PFA fixation optimally preserves pancreas cell morphology and improves staining quality. Tissues may be embedded in either Optimal Temperature Cutting (OCT) media or carboxymethylcellulose (CMC).

Prepare coverslips with pancreas sections.

Protocol
CODEX® Multiplexed Imaging | Tissue Sectioning
NAME

CODEX® Multiplexed Imaging | Tissue Sectioning

CREATED BY
Islet and Pancreas Analysis Core

ⓘ Samples mounted on coverslips can be stored at Temperature-80 °C for up to 2 months. All of our optimization of this protocol was done on Thikness10 µm cryosections.

Design antibody panel using a combination of preconjugated or custom-conjugated primary antibodies.

Protocol
CODEX® Multiplexed Imaging | Antibody Conjugation and Validation
NAME

CODEX® Multiplexed Imaging | Antibody Conjugation and Validation

CREATED BY
Islet and Pancreas Analysis Core

ⓘ For information on HuBMAP Organ Mapping Antibody Panels (OMAPs), please visit the CCF Portal here.

Label tissue with primary antibodies and prepare reagents for imaging.
Protocol
CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation 
NAME

CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation 

CREATED BY
Islet and Pancreas Analysis Core

ⓘ See also: Akoya Biosciences PhenoCycler™ User Manual

Perform cyclic fluorescent imaging of tissue.
Protocol
CODEX® Multiplexed Imaging | Microscope Setup and Tissue Imaging
NAME

CODEX® Multiplexed Imaging | Microscope Setup and Tissue Imaging

CREATED BY
Islet and Pancreas Analysis Core

Process raw imaging data.

As of May 2022, raw imaging data is processed using CODEX Processor v1.8.1.9 (Akoya Biosciences), which performs image stitching, z-plane selection, background subtraction, deconvolution, and shading correction.

ⓘ See also: CODEX® Processor User Instructions & Technical Notes

[Optional] Perform registration with other imaging modalities.

Autofluorescence (AF) Registration (HuBMAP)
Protocol
Data Processing and Image Registration
NAME

Data Processing and Image Registration

CREATED BY
Jamie Allen