Human Nephrin Antibody

Detection of Human Nephrin by Western Blot. Western blot shows lysates of human kidney tissue under reducing and non-reducing conditions. PVDF membrane was probed with 1 µg/mL Sheep Anti-Human Nephrin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4269) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band for Nephrin was detected at approximately 150 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

Nephrin in Human Kidney. Nephrin was detected in immersion fixed paraffin-embedded sections of human kidney using 1.7 µg/mL Sheep Anti-Human Nephrin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4269) overnight at 4 °C. Tissue was stained with the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific labeling was localized to podocytes in glomeruli. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human Nephrin by Simple Western^TM. Simple Western lane view shows lysates of human kidney, loaded at 0.2 mg/mL. A specific band was detected for Nephrin at approximately 185 kDa (as indicated) using 20 µg/mL of Sheep Anti-Human Nephrin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4269) followed by 1:50 dilution of HRP-conjugated Anti-Sheep IgG Secondary Antibody (HAF016). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human Nephrin by Immunocytochemistry/Immunofluorescence Concordant expression of developmental programs across organoids from four human iPSC lines. a Heatmap of expression patterns for major nephrogenesis markers across organoid differentiation time points (iPSC D0, D7, D15, and D29, averaged across four cell lines, ML protocol) and human adult kidney. Expression values were row-normalized to obtain z-scores; red color indicates positive z-scores. b Canonical (NPHS2) and data-derived (CLDN5) podocyte marker genes superimposed in tSNE plots from D15 organoids (N2 line, ML protocol). c IF staining of D15 kidney organoid (N2 line, ML protocol) for CLDN5 as a marker of early podocyte differentiation derived from the single-cell data. Additional canonical podocyte markers (NPHS1, WT1) and DAPI staining as shown. d IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31784515), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Nephrin by Immunocytochemistry/Immunofluorescence Organoid Glomeruli model of congenital nephrotic syndrome in vitro. a Description of the NPHS1 variants identified in the patient modelled, diagnosed with congenital nephrotic syndrome (CNS). b–d Immunostaining of OrgGloms isolated from control organoids and CNS patient organoids show reduced NEPHRIN and PODOCIN protein levels in the organoids derived from patient-iPSC, representative images shown of >3 biological replicates. Scale bars 10 µm. e Higher power immunofluorescent images show the polarised co-localisation of NEPHRIN with NEPH1 (solid white arrowheads) and PODOCIN in control OrgGloms. This polarisation is lost in CNS OrgGloms due to the absence of NEPHRIN (white arrows). Scale bars 10 µm. f Quantitative analysis of fluorescence intensities from independent OrgGlom biological replicates performed using one control and two distinct patient-derived CNS iPSC clones. Organoid glomeruli generated from both patient-derived iPSC clones show significant reduction in NEPHRIN and PODOCIN protein levels. Two-way ANOVA p < 0.0001; error bars = SEM. Biological replicates. NEPHRIN (controls, n = 20; CNS, n = 56); PODOCIN (controls, n = 14; CNS, n = 22); CD2AP (controls, n = 8; CNS, n = 15); NEPH1 (controls, n = 10; CNS, n = 17). Significant difference assessed by Sidak’s multiple comparisons test between cell lines; F-value = 112; DF = 1. NEPHRIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p > 0.9999. PODOCIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p = 0.9995. CD2AP: control vs CNS#1, p = 0.0007; control vs CNS#2, p = 0.0016; CNS#1 vs CNS#2, p = 0.9980. NEPH1: control vs CNS#1, p = 0.5320; control vs CNS#2, p = 0.9994; CNS#1 vs CNS#2, p = 0.9992. g Quantitative western blot analysis of NEPHRIN and PODOCIN protein levels within independent biological replicates confirms the significant depletion of these proteins in OrgGloms derived from CNS iPSCs Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30514835), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Nephrin by Immunohistochemistry IF validation of markers derived from the single-cell data in mature kidney organoids. a IF staining of an entire kidney organoid with segment specific markers as shown. b Schematic of kidney nephron with major cell types and canonical markers annotated. c Immunofluorescence staining of D29 kidney organoids for podocyte (WT1), proximal tubule (LTL), and distal tubule (CDH1 and GATA3) across two protocols (JB, ML) and four cell lines (AS, N1, N2, ThF). IF staining for validation of markers identified in the single-cell data: d NPHS1 colocalized with the podocyte-specific marker SYNPO and e LRP2 colocalized with the proximal tubular marker LTL (bottom). f IF staining validation for MEIS1-positive mesenchymal cells in D29 organoids. LAMA1 indicates basement membranes. MEIS1 staining of mesenchymal cells appropriately surrounds LAMA1-defined tubular nephron structures. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31784515), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Nephrin by Immunocytochemistry/Immunofluorescence Organoid Glomeruli model of congenital nephrotic syndrome in vitro. a Description of the NPHS1 variants identified in the patient modelled, diagnosed with congenital nephrotic syndrome (CNS). b–d Immunostaining of OrgGloms isolated from control organoids and CNS patient organoids show reduced NEPHRIN and PODOCIN protein levels in the organoids derived from patient-iPSC, representative images shown of >3 biological replicates. Scale bars 10 µm. e Higher power immunofluorescent images show the polarised co-localisation of NEPHRIN with NEPH1 (solid white arrowheads) and PODOCIN in control OrgGloms. This polarisation is lost in CNS OrgGloms due to the absence of NEPHRIN (white arrows). Scale bars 10 µm. f Quantitative analysis of fluorescence intensities from independent OrgGlom biological replicates performed using one control and two distinct patient-derived CNS iPSC clones. Organoid glomeruli generated from both patient-derived iPSC clones show significant reduction in NEPHRIN and PODOCIN protein levels. Two-way ANOVA p < 0.0001; error bars = SEM. Biological replicates. NEPHRIN (controls, n = 20; CNS, n = 56); PODOCIN (controls, n = 14; CNS, n = 22); CD2AP (controls, n = 8; CNS, n = 15); NEPH1 (controls, n = 10; CNS, n = 17). Significant difference assessed by Sidak’s multiple comparisons test between cell lines; F-value = 112; DF = 1. NEPHRIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p > 0.9999. PODOCIN: control vs CNS#1, p < 0.0001; control vs CNS#2, p < 0.0001; CNS#1 vs CNS#2, p = 0.9995. CD2AP: control vs CNS#1, p = 0.0007; control vs CNS#2, p = 0.0016; CNS#1 vs CNS#2, p = 0.9980. NEPH1: control vs CNS#1, p = 0.5320; control vs CNS#2, p = 0.9994; CNS#1 vs CNS#2, p = 0.9992. g Quantitative western blot analysis of NEPHRIN and PODOCIN protein levels within independent biological replicates confirms the significant depletion of these proteins in OrgGloms derived from CNS iPSCs Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30514835), licensed under a CC-BY license. Not internally tested by R&D Systems.