Human Podocalyxin Antibody

Detection of Human Podocalyxin by Western Blot. Western blot shows lysates of human kidney tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Podocalyxin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1658) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for Podocalyxin at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Podocalyxin in Human Kidney. Podocalyxin was detected in immersion fixed paraffin-embedded sections of human kidney using 5 µg/mL Goat Anti-Human Podocalyxin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1658) overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) 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.

Western Blot Shows Human Podocalyxin Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and Podocalyxin/PODXL knockout HeLa cell line (KO). PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Podocalyxin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1658) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Podocalyxin at approximately 160 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Podocalyxin Like by Immunocytochemistry/Immunofluorescence Podocalyxin promotes lumenogenesis in epiblast spheroids.(a) Immunoblot for podocalyxin protein in two representative PODXL−/− hPSC mutant clones (m1 and m2), compared with CRISPR/Cas9 non-mutant wild-type clones (WT) or cells subjected to scrambled (scr) or podocalyxin (pod) siRNA knockdown. (b) Brightfield images of sandwiched parental ESCs were compared with two mutant or two WT CRISPR/Cas9 clones. (c) Cavitated spheroids as a percentage of total colonies. Data from pools of WT or mutant cell lines were averaged to determine group means (AVG, n≥9) and P values. (d) Podocalyxin and ZO-1 immunofluorescence in naive and primed hLR5 hPSCs or (e) mESCs and EpiSCs. (f) Confocal z-sections of undifferentiated hPSCs showing localization of ZO-1 and beta CAT in unmodified (WT) or PODXL−/− colonies. (g) Filamentous actin (f-actin) and occludin (OCLN) immunofluorescence in undifferentiated WT or PODXL−/− clones. (h) Averaged TEER measurements in WT or PODXL−/− monolayers (n≥3). Scale bars, 50 μm or (f,g) 20 μm. Error bars, s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26493500), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Podocalyxin Like by Immunocytochemistry/Immunofluorescence Podocalyxin promotes lumenogenesis in epiblast spheroids.(a) Immunoblot for podocalyxin protein in two representative PODXL−/− hPSC mutant clones (m1 and m2), compared with CRISPR/Cas9 non-mutant wild-type clones (WT) or cells subjected to scrambled (scr) or podocalyxin (pod) siRNA knockdown. (b) Brightfield images of sandwiched parental ESCs were compared with two mutant or two WT CRISPR/Cas9 clones. (c) Cavitated spheroids as a percentage of total colonies. Data from pools of WT or mutant cell lines were averaged to determine group means (AVG, n≥9) and P values. (d) Podocalyxin and ZO-1 immunofluorescence in naive and primed hLR5 hPSCs or (e) mESCs and EpiSCs. (f) Confocal z-sections of undifferentiated hPSCs showing localization of ZO-1 and beta CAT in unmodified (WT) or PODXL−/− colonies. (g) Filamentous actin (f-actin) and occludin (OCLN) immunofluorescence in undifferentiated WT or PODXL−/− clones. (h) Averaged TEER measurements in WT or PODXL−/− monolayers (n≥3). Scale bars, 50 μm or (f,g) 20 μm. Error bars, s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26493500), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Podocalyxin Like by Immunocytochemistry/Immunofluorescence Junctional complexes are disrupted in hPSC-derived PODXL−/− podocyte-like cells.(a) Confocal optical section of adult human kidney. Podocalyxin is expressed in podocytes and peritubular capillaries, but is absent from tubules (white dotted lines). Auto, autofluorescence. (b) Crumbs3 expression in hPSC-derived kidney organoids (confocal red channel) and human kidney tissue (far right panel, immunohistochemistry). (c) Confocal optical sections showing distributions of ZO-1 with podocalyxin or (d) beta CAT in hPSC-derived podocyte-like cell clusters. Arrowheads highlight tracks of junctional complexes between podocyte-like cells. (e) Confocal sections of wild-type or PODXL−/− podocyte-like cell clusters in tubular organoids. (f) Gap widths between adjacent podocyte-like cell nuclei in these cell lines (n≥100 gaps pooled from two experiments). Scale bars, 50 μm. Error bars, s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26493500), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Podocalyxin Like by Immunocytochemistry/Immunofluorescence Junctional complexes are disrupted in hPSC-derived PODXL−/− podocyte-like cells.(a) Confocal optical section of adult human kidney. Podocalyxin is expressed in podocytes and peritubular capillaries, but is absent from tubules (white dotted lines). Auto, autofluorescence. (b) Crumbs3 expression in hPSC-derived kidney organoids (confocal red channel) and human kidney tissue (far right panel, immunohistochemistry). (c) Confocal optical sections showing distributions of ZO-1 with podocalyxin or (d) beta CAT in hPSC-derived podocyte-like cell clusters. Arrowheads highlight tracks of junctional complexes between podocyte-like cells. (e) Confocal sections of wild-type or PODXL−/− podocyte-like cell clusters in tubular organoids. (f) Gap widths between adjacent podocyte-like cell nuclei in these cell lines (n≥100 gaps pooled from two experiments). Scale bars, 50 μm. Error bars, s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26493500), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Podocalyxin Like by Immunocytochemistry/Immunofluorescence hPSCs form cavitated spheroids in 3D culture.(a) Schematic of spheroid-to-organoid culture protocol. (b) Phase contrast images of ESCs in sandwich (3D) or monolayer (2D) cultures. Consecutive days are shown, with d0 indicating the time point immediately before sandwiching. (c) Confocal optical sections showing PODXL, ZO-1 and beta CAT immunofluorescence through a representative spheroid with cavity. Vertical distance from top to bottom row is shown at left. (d) Representative brightfield images of hPSCs in 3D cultures that were dissociated (coloured frames) and passaged (matching coloured arrows). Dashed arrows represent serial passages in the 3D condition. Lower row shows cells plated into 2D cultures from dissociated spheroids from each passage. (e) Hematoxylin and eosin-stained sections of teratomas generated from hPSC serial 3D passages p3, p6 and p9 showing pigmented epithelium (ectoderm), cartilage (mesoderm) and glandular epithelium (endoderm). (f) Cell number (average of duplicate counts for each time point, or AVG of all five time points shown in the last column) in 2D and 3D cultures 72 h after plating. (g) Representative immunofluorescence images showing OCT4 and sex-determining region Y box-2 (SOX2) or tumor rejection antigen 1–60 (TRA-1-60) and NANOG localization in p3 and p7 serially sandwiched hPSCs. Scale bars, 100 μm. Error bars, s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26493500), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Podocalyxin Like by Immunocytochemistry/Immunofluorescence Junctional complexes are disrupted in hPSC-derived PODXL−/− podocyte-like cells.(a) Confocal optical section of adult human kidney. Podocalyxin is expressed in podocytes and peritubular capillaries, but is absent from tubules (white dotted lines). Auto, autofluorescence. (b) Crumbs3 expression in hPSC-derived kidney organoids (confocal red channel) and human kidney tissue (far right panel, immunohistochemistry). (c) Confocal optical sections showing distributions of ZO-1 with podocalyxin or (d) beta CAT in hPSC-derived podocyte-like cell clusters. Arrowheads highlight tracks of junctional complexes between podocyte-like cells. (e) Confocal sections of wild-type or PODXL−/− podocyte-like cell clusters in tubular organoids. (f) Gap widths between adjacent podocyte-like cell nuclei in these cell lines (n≥100 gaps pooled from two experiments). Scale bars, 50 μm. Error bars, s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26493500), licensed under a CC-BY license. Not internally tested by R&D Systems.