Elsevier

Kidney International

Volume 101, Issue 1, January 2022, Pages 137-143
Kidney International

Technical Notes
Highly multiplexed immunofluorescence of the human kidney using co-detection by indexing

https://doi.org/10.1016/j.kint.2021.08.033Get rights and content

The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

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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