Ab207170

The immunohistochemical procedure was provided in the recent reports.^[37, ³⁸, ³⁹, ⁴⁰, ^41] Briefly, monolayer cells and kidney organoids in cell culture flasks were fixed in 4% paraformaldehyde for 20 min at 4 °C, followed by 3 PBS washes. Samples were blocked with 5% goat serum in 0.1% Triton X‐100/PBS for 2–3 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The following antibodies and dilutions were used: goat anti‐nephrin (1:200, R&D AF3159), rabbit anti‐NCC (1:100, StressMarq SPC‐402), rabbit anti‐CD31 (1:200, Abcam ab28364), rabbit anti‐podocin (1:200, Abcam ab50339), rabbit anti‐Six2 (1:200, Proteintech #11562‐1‐AP), goat anti‐GATA‐3 (1:200, R&D AF2605‐SP), mouse anti‐E‐cadherin (1:200, Invitrogen #13‐1900), rabbit anti‐cleaved‐Notch1 (1:100, Cell Signaling #4147S), rabbit anti‐UMOD (1:100, Abcam ab207170), and LTL‐fluorescein (1:1000, Vector Laboratories FL‐1321). After 3 10‐min washes with 0.1% Triton X‐100/PBS, samples were incubated with secondary antibodies for 1 h at room temperature. Corresponding Alexa Fluor 448, 555, or 647 secondary antibodies (Invitrogen) were used at 1:1000. Images were captured using an Olympus IX73 fluorescence microscope (Japan).

The antibodies used for Western blot and immunohistochemistry (IHC) included rabbit monoclonal anti-uromodulin antibody (ab207170, Abcam, Cambridge, UK), mouse monoclonal anti-Factor H antibody (ab118820, Abcam, UK), mouse monoclonal anti-alpha-smooth muscle actin (α-SMA) (ab7817, Abcam, UK), mouse anti-uromodulin antibody (sc-271022, Santa Cruz Bio., Dallas, TX, USA), mouse monoclonal anti-GAPDH antibody (ab8245, Abcam, UK), and rabbit monoclonal anti-C3 antibody (ab200999, Abcam, UK). Secondary antibodies for immunoblotting were horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H + L) (ZB-2305, ZSGB-BIO, Beijing, China) and mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Bio., Dallas, TX, USA).