Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry

Singh, Nikhil; Avigan, Zachary M.; Kliegel, Judith A.; Shuch, Brian; Montgomery, Ruth R.; Moeckel, Gilbert; Cantley, Lloyd G.

doi:10.1172/jci.insight.129477

Cited by 41 publications

(43 citation statements)

References 53 publications

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“…Analyses of various substructures in the renal cortex revealed their spatial relationships. Composites of pseudocolored images revealed a predominant presence of proximal tubules (AQP1) consistent with the histology of renal cortex ( Figure 8A ) (64, 65). We were also able to identify the collecting duct (AQP2+ AQP3+ EpCAM+) (Cyto7+ Cyto8+) ( Figure 8A and 8C ) (66–68), Distal convoluted tubules (AQP2-AQP3-EpCAM+), Thick Ascending Loop of Henle’s (EpCAM+ Uromodulin+) and their spatial location with respect to one another ( Figure 8A ) (69).…”

Section: Resultssupporting

confidence: 74%

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

Rajagopalan

Venkatesh

Aslam

et al. 2020

Preprint

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Spatial organization of molecules and cells in complex tissue microenvironments provides essential cues during healthy growth and in disease. Novel techniques are needed for elucidation of their spatial relationships and architecture. Although a few multiplex immunofluorescence based techniques have been developed for visualization of the spatial relationships of various molecules in cells and tissues, there remains a significant need for newer methods that are rapid, easy to adapt and are gentle during the cyclic steps of fluorescence staining and de-staining. Here, we describe a novel, multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA labelled antibodies for immunofluorescence staining of cells and tissues, and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies and antibody fragments, such as Fabs, to efficiently analyse complex cells and tissues in multiple rounds of staining and de-staining. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals, while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy to use and gentle new technique for spatialomic analyses of complex microenvironments.

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Section: Resultssupporting

confidence: 74%

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

Rajagopalan

Venkatesh

Aslam

et al. 2020

Preprint

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“…Thus, IMC offers a major advantage over classical immunohistochemistry techniques which are limited by the numbers of markers that can be included simultaneously. Together with other studies that have developed antibody panels for FFPE tissue (15,21,22) this sets the stage for detailed studies to determine immune heterogeneity and cell-cell interactions in situ, providing a novel layer of understanding of functioning of the immune system on tissues. We anticipate that our study will guide other researchers that wish to use IMC for analysis of tissue of choice.…”

Section: Discussionmentioning

confidence: 99%

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

et al. 2020

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Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantification in situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at −20 • C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at −20 • C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4 • C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of α-smooth muscle actin (α-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality.

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“…For each participant, an 18-gauge core or wedge biopsy was collected intraoperatively during organ preparation and processed as previously described. 28 …”

Section: Methodsmentioning

confidence: 99%

“… 27 Our group has previously developed and validated an antibody panel defining the cellular diversity of reference human kidney tissue and an automated kidney-specific data analysis pipeline. 28 …”

Section: Introductionmentioning

confidence: 99%

Tubular Cell Dropout in Preimplantation Deceased Donor Biopsies as a Predictor of Delayed Graft Function

Avigan

Singh

Kliegel

et al. 2021

Transplantation Direct

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Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry

Cited by 41 publications

References 53 publications

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

Tubular Cell Dropout in Preimplantation Deceased Donor Biopsies as a Predictor of Delayed Graft Function

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