Peroxidazed1 solution

Duboscq-Brazil-fixed, paraffin-embedded renal sections (3 μm) were deparaffinized and incubated with Peroxidazed1 solution (Biocare Medical) to quench endogenous peroxidases. Antigen retrieval was performed as described above. After blocking with Background Punisher (Biocare Medical), sections were incubated with rabbit anti-Ki67 (Abcam), goat anti-CD144 (1:100), rabbit anti-CD31 (1:150), or rabbit anti-ZO-1 (1:200), followed by MACH4 or goat-on-rodent-HRP Polymer kit (Biocare Medical) and diaminobenzidine substrate solution. Slides were finally counterstained with Meyer’s hematoxylin, dehydrated in graded alcohols and observed by light microscopy (Apotome Axio Imager Z2, Zeiss). Isotype control staining was used to exclude the nonspecific binding (normal rabbit IgG, Santa Cruz Biotechnology). Glomerular repopulation was quantified as the percentage of glomeruli reached by iPSC-derived ECs of the total number of glomeruli observed (100%). At least 1200 glomeruli were examined on ZO-1-stained sections. Glomeruli were considered fully repopulated when the iPSC-derived EC repopulation involved 75% to 100% of the glomerular tuft. When the repopulation involved less than 75% of the glomerular area, glomeruli were considered partially repopulated. Data are expressed as mean ± SD.

Paraffin blocks were sectioned using a microtome to obtain 4 μm thick sections for immunostaining. The paraffin sections were dewaxed in xylene and hydrated in decreasing concentrations of ethanol. Sections were then incubate in 1 × DIVA Decloaker antigen retrieval solution (Biocare Medical) at 110°C for 15 min using the decloaking chamber (Biocare Medical). Following antigen retrieval, sections were incubated in peroxidazed 1 solution (Biocare Medical) at room temperature for 5 min to quench endogenous peroxidase activity. After blocked with background sniper at room temperature for 10 min, sections were incubated with a monoclonal rabbit anti-human CD3 antibody (0.3 μg/ml; Biocare Medical) in Dako REAL antibody diluent (Dako) at room temperature for 1 h. Sections were subsequently incubated with HRP-labeled goat anti-rabbit IgG polymer (Dako) at room temperature for 30 min. Finally, sections were exposed to liquid DAB+ substrate chromogen system (Dako) at room temperature for 5 min and counterstaining was performed using Gill's hematoxylin (Sigma).