May 25, 2022
Version 1
CODEX® Multiplexed Imaging | Modality Overview V.1
- Diane Saunders1,
- Conrad Reihsmann2,
- Alexander Hopkirk2,
- Marcela Brissova1,
- Alvin C. Powers1
- 1Vanderbilt University, Vanderbilt University Medical Center;
- 2Vanderbilt University Medical Center
- Vanderbilt Diabetes Research Center
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 26, 2022
Last Modified: May 25, 2022
Protocol Integer ID: 59918
Keywords: multiplexed imaging of the human pancrea, performing multiplexed imaging, use by the vanderbilt diabetes research center islet, vanderbilt diabetes research center islet, pancreas analysis, vanderbilt diabetes research center, human pancrea
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).
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
CREATED BY
Islet and Pancreas Analysis Core
ⓘ Samples mounted on coverslips can be stored at -80 °C for up to 2 months. All of our optimization of this protocol was done on 10 µm cryosections.
Protocol
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
CREATED BY
Islet and Pancreas Analysis Core
ⓘ See also: Akoya Biosciences PhenoCycler™ User Manual
Perform cyclic fluorescent imaging of tissue.
Protocol
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.
[Optional] Perform registration with other imaging modalities.
Autofluorescence (AF) Registration (HuBMAP)