CODEX® Multiplexed Imaging | Modality Overview
Diane Saunders, Marcela Brissova, Alvin C. Powers, Conrad Reihsmann, Alexander Hopkirk
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.
Steps
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)
Prepare coverslips with pancreas sections.
CODEX® Multiplexed Imaging | Tissue Sectioning
ⓘ 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.
Design antibody panel using a combination of preconjugated or custom-conjugated primary antibodies. preconjugated or custom-conjugated primary antibodies.
CODEX® Multiplexed Imaging | Antibody Conjugation and Validation
ⓘ 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.
CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation
ⓘ See also: Akoya Biosciences PhenoCycler™ User Manual
Perform cyclic fluorescent imaging of tissue.
CODEX® Multiplexed Imaging | Microscope Setup and Tissue Imaging
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)
