Ab39250

Based on the previously reported methods [26 ], the immunofluorescence staining was conducted to assess the expressions of TJPs claudin5, occludin, and ZO-1. The primary antibodies were anti-claudin5 (Abcam, Assay dependent, ab15106), anti-occludin (Abcam, 1:100 dilution, ab216327), anti-ZO-1 (Abcam, 1:100 dilution, ab221547), anti-VEGFA (Abcam, 1 µg/mL, ab39250), and anti-MMP-9 (Abcam, 1:500 dilution, ab76003). The secondary antibodies were Alexa Flur 488 mouse anti-rabbit IgG and tetraethyl rhodamine isothiocyanate goat anti-rabbit IgG. The nuclei were stained using 4′,6-Diamidino-2-phenylindole staining solution (Sigma-Aldrich). Ultimately, Vectra® Polaris™ Automated Quantitative Pathology Imaging System (PerkinElmer, USA) was used to analyze all images.

hDPSCs cultured in 24-well plates were fixed with 4% PFA in PBS for 20 min at room temperature. After washing in PBS, samples were permeabilized with 0.1% Triton X-100 for 5 min and blocked with 1% BSA in PBS. Incubation with primary anti-OC antibody (sc-30044 Santa Cruz) was performed overnight at 4°C. Primary antibody was revealed using a FITC-conjugated anti-rabbit IgG secondary antibody. Nuclei were stained with Hoechst.
Frozen sections were stained by Hematoxylin and Eosin or permeabilized with 0.1% Triton X-100 for 15 min. Incubation with primary antibodies anti-OPN (ab8448 Abcam, Cambridge, UK), anti-OC (sc-30044 Santa Cruz), and anti-VEGF (ab39250 Abcam Cambridge, UK) was performed overnight at 4°C. FITC conjugated secondary antibody to rabbit IgG was used to reveal primary antibodies. Micrographs were taken with a microscope EVOS FL Cell Imaging System (Life Technologies).

PI3K (mouse anti-mouse anti-PI3 kinase antibody, 1:150, ab140307, Abeam, UK), AKT (rabbit anti-rabbit anti-AKT1 + AKT2 + AKT3 antibody, 1:250, ab179463, Abeam, UK), and VEGFA (rabbit anti-rabbit anti-VEGF antibody, 1:20000, ab39250, Abcam, UK) were used in immunohistochemistry.
We selected 10 renal biopsy samples, including 8 IMN and 2 normal renal tissue samples (Supplementary Table 1). Serum anti-PLA2R levels were significantly increased in 8 patients with IMN.
Renal tissue samples were fixed in 4% paraformaldehyde, rinsed with tap water, stained with a SABC immunohistochemistry kit, developed with diaminobenzidine (DAB), dehydrated with a graded series of ethanol, cleared with xylene and sealed with neutral gum. Finally, the renal sections were observed under a 200 × optical microscope (Olympus, Japan). Quantification of target protein staining was performed only on glomeruli using an IHC profiler in Image-Pro Plus, in which the intensity of the staining was quantified.