Immunohistochemical Analysis of Kidney Samples

Cell clusters or excised kidneys were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Science; 15,714) for 1 h at room temperature (RT) or overnight at 4 °C, respectively, before being paraffin embedded and sectioned at 10 μm. Paraffin was removed with Histoclear (Thermo Scientific; C78-2-G) and rehydrated, and antigens retrieved by 2 h 0.1 M EDTA (Ambion; AM9261) treatment in a pressure cooker (Proteogenix; 2,100 Retreiver). Samples were blocked with 0.1% Triton X-100 (VWR; EM-9400) and 5% donkey serum (Jackson Immunoresearch; 017-000-121) in PBS (staining solution) for 1 h at RT, incubated with primary antibodies diluted in staining solution overnight at 4 °C, washed for 5 min in staining solution, incubated covered with appropriate Alexa Fluor-488 or -594 secondary antibodies diluted 1:300 in staining solution 2 h at RT, washed for 5 min in staining solution, mounted with Vectashield (Vector Laboratories; H-1200) and covered with a coverslip. Images were taken with an Olympus IX51 Microscope. The primary antibodies used to assess differentiation throughout this study are given in Supplementary Table 6 (ref. 17 (link)).