Anti podocalyxin

The following primary anti-human antibodies were used: sheep anti-podoplanin, goat anti- VEGFR-3, anti-LYVE-1, anti-PROX1, anti-ITGA9, anti-VEGFR-2, anti-NRP2, anti-podocalyxin and anti-GFP, all from R&D Systems, Minneapolis, MN. Mouse anti-human CD14 and anti-CD68 antibodies were from Santa Cruz Biotechnology, Dallas, TX and Thermo Fisher, Waltham, MA, respectively. Rabbit anti-acetylated histone H3 and anti-Ki-67 were from Upstate, Billerica, MA and Cell Signaling Technologies, Danvers, MA, respectively. Rabbit anti-mouse Lyve-1 and rat anti-Meca-32 antibodies were from AngioBio, Del Mar, CA, and BioXCell, West Lebanon, NH, respectively. Secondary antibodies conjugated to FITC, Cy3, DyLight 488, DyLight 549, and APC donkey anti-rabbit, anti-sheep, anti-rat, anti-mouse and anti-goat IgG were all from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cryosections with a thickness of 4 μm were prepared using a cryostat and were fixed in 4% paraformaldehyde for 15 min. After blocking, the cryosections were incubated with primary antibodies and then with a fluorescein Cy3-FITC-labelled secondary antibody (1:100; Proteintech, Wuhan, China). Fluorescence images were recorded using a TCS SP5II confocal microscope (Leica, Bensheim, Germany). The following primary antibodies were used: anti-desmin (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-podocalyxin (1:100; R&D Systems, Minneapolis, MN, USA), and anti-snail2 (1:100, Proteintech). Podocytes were seeded onto clean glass coverslips, fixed with 4% paraformaldehyde, and permeabilised with 0.2% Triton X-100. The slides were incubated with an anti-PCNA (1:100, Proteintech), anti-synaptopodin (1:100; Proteintech), anti-CDK4 (1:100; Abcam, Cambridge, UK), anti-P-YAP (1:100; Cell Signaling Technology, Danfoss, MA, USA), or anti-snail2 (1:100, Proteintech) antibody.
For immunohistochemistry analysis, after deparaffinisation, rehydration, antigen retrieval, and blocking, the sections were incubated with an anti-PCNA (1:100), anti-CDK4 (1:100), anti-desmin (1:100), anti-YAP (1:100, Proteintech), or anti-cyclin D1 (1:100, Cell Signaling Technology) primary antibody and then with a horseradish peroxidase-labelled secondary antibody (Beyotime, Shanghai, China).

Mouse ECs transduced with lentiviral vectors carrying shRNA for Nova2 were seeded in 35-mm Petri dishes coated with Gelatin (Difco) 0.1% and cultured for 72 h. The splitting ratio was such that confluence was reached overnight after seeding. For immunofluorescence, ECs were fixed with 4% paraformaldehyde (PFA) and then permeabilized with 0.5% Triton X-100 for 10 min. Blocking (1 h), primary (overnight) and secondary (1 h) antibodies were diluted in PBS with 2% BSA. The following primary antibodies were used: anti-Par3 (1:100 Millipore), anti-Podocalyxin (1:100 R&D), anti-VE-cadherin (1:100 C-19, sc-6458, goat, Santa Cruz Biotechnology) and anti-β-catenin (1:50 BD Transduction Laboratories). Secondary antibodies for immunofluorescence were donkey antibodies to the appropriate species conjugated with Alexa Fluor 488, 555 or 647 (dilution 1:200 or 1:400).
For imaging, charge-coupled device camera on epifluorescence microscope (Leica) or Leica TCS SP2 confocal microscopy were used. ImageJ (NIH) was employed for data analysis. Figures were assembled using Adobe Photoshop and Adobe Illustrator. Only adjustments of brightness and contrast were used in the preparation of the figures. For comparison purposes, different sample images of the same antigen were acquired under constant acquisition settings.

Embryoid bodies or kidney organoids were fixed with paraformaldehyde (4%) in PBS for 1 h. Immunofluorescence was performed as previously described (Morizane et al., 2015 (link)). The primary antibodies used were anti-GATA4 (Santa Cruz, #SC-1237), anti-FOXF1 (R&D systems, #AF4798), anti-β III tubulin (Millipore, #MAB1637), anti-OAT1, anti-OAT3, anti-OCT2, anti-KIM1, anti-γH2AX (Cell Signaling, #2577), anti-nephrin (Progen Biotechnik, GP-N2), anti-LTL, anti-podocalyxin (R&D systems, #1658), and Phalloidin (Molecular Probes, #R-415). Alexa 488, 555, or 647 dye-labeled (Molecular Probes; Invitrogen) secondary antibodies were used for immunofluorescence. For 3D whole-mount immunohistochemistry, kidney organoids underwent an additional clearing process after immunostaining as previously described (Hiratsuka et al., 2019 (link)). Immunofluorescence images were obtained using C1 confocal (Nikon) or Stellaris 8 (Leica).

HUVECs were transduced with GIPZ lentiviral vectors (Open Biosystems) carrying shRNA for Nova2 or control shRNA. Control and Nova2 knockdown HUVECs were cultured in 3D collagen gel. The final cell density in collagen (3.5 mg ml⁻¹ final concentration collagen type1 from rat tail, High Concentration, BD Biosciences) was 5 × 10⁵ cells ml⁻¹. Culture medium was 199 with 1% FCS, Insulin-Transferrin-Selenium supplement (Life Technologies), 50 ng ml⁻¹ phorbol myristate acetate, 50 μg ml⁻¹ ascorbic acid, 40 ng ml⁻¹ VEGF and 40 ng ml⁻¹ bFGF. For confocal microscopy, 190 μl cell suspension in collagen was used for each microwell (μ-slide 80826, Ibidi, Germany). 3D cultures were fixed with 3% PAF for 35min, quenched with 75mM NH₄Cl and 20mM glycine in PBS, pH 8, for 10min and blocked with 0.7% FSG and 0.3% Triton X-100 PBS (blocking buffer) for 30min. Primary and secondary antibodies were incubated overnight at 4°C. Primary antibody contained 5% donkey serum. Washes in blocking buffer were performed over the course of a day at room temperature. For immunofluorescence, the following primary antibodies were used: anti-Podocalyxin (R&D, 1:400) and anti-Coll IV (AbD Serotech, 1:200). Secondary antibodies for immunofluorescence were donkey antibodies to the appropriate species conjugated with Alexa Fluor 488, 555 or 647 (dilution 1:200 or 1:400).