Technical NotesHighly multiplexed immunofluorescence of the human kidney using co-detection by indexing
Graphical abstract
Section snippets
Methods
Normal portions of kidney cancer nephrectomies from adult patients were studied in addition to kidneys from diabetic nephropathy patients. Tissue blocks were frozen over an isopentane dry ice slurry, and 10-μm sections were thaw mounted onto glass cover slips. These sections were then fixed and incubated with a mixture containing all primary antibodies. Secondary oligonucleotide sequences were automatically added and removed serially for multiplexed visualization on a single section without
Multiplexed IF imaging using CODEX
We have generated CODEX multiplexed IF images from human kidney tissue using 23 barcoded antibodies (Table 1). The major structures within the kidney were imaged, such as the endothelial layer within glomeruli, proximal tubules, and collecting ducts, by targeting cluster of differentiation (CD) 93, aquaporin 1, and calbindin, respectively. These structures are further visually subdivided by targeting antigens that localize to different tubules or tubular layers (e.g., endothelium [CD90],
Discussion
Using 23 antibodies, key kidney cell types can be visualized and compared in human kidney tissue. This study incorporates a combination of commercial CODEX labels as well as conjugated purified antibodies from 3 different vendors, demonstrating the flexibility of the approach to accommodate a variety of antibody sources. Our studies support that well-validated, primary antibodies free from common preservations (e.g., bovine serum albumin, glycerol, and sodium azide) are compatible with this
Disclosure
All the authors declared no competing interests.
Data Availability Statement
The data supporting the findings of this study are openly available at the National Institutes of Health Human Biomolecular Atlas Program (HuBMAP) data portal at https://portal.hubmapconsortium.org.
Acknowledgments
Support was provided by the National Institutes of Health (NIH) Common Fund and National Institute of Diabetes and Digestive and Kidney Diseases (U54DK120058 awarded to JMS and RMC) and NIH National Institute of Allergy and Infectious Disease (R01AI138581 awarded to JMS). EKN is supported by a National Institute of Environmental Health Sciences training grant (T32ES007028). Human tissues were acquired through the Cooperative Human Tissue Network at Vanderbilt University Medical Center, which is
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