Fluoroshield mounting medium containing 4 6 diamidino 2 phenylindole dapi

Immunofluorescence staining was performed by following the method described previously [53 (link)]. Briefly, kidneys were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan), and frozen in liquid nitrogen. Sections of 5-μm thickness were cut by a cryostat (CM3050; Leica, Wetzlar, Germany); then incubated with primary antibodies against type I collagen (Acris Antibodies, Germany), type IV collagen, type V collagen, synaptopodin, VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), nephrin, and podocalyxin (R&D Systems, Minneapolis, MN, USA), respectively, at 4 °C overnight, and followed by secondary antibodies conjugated to Alexa Fluor^® 488 or 568 (Invitrogen, Carlsbad, CA, USA); double-stained with rhodamine-conjugated phalloidin (Life Technologies, Gaithersburg, MD, USA) for F-actin; and finally submerged in fluoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, UK). Confocal imaging was performed according to the method described previously [26 (link)] with a confocal microscope (LSM700; Carl Zeiss, Jena, Germany). Alexa Fluor^® 488, and 568 signals were detected at laser excitation wavelengths of 488 nm and 543 nm, respectively.

After deparaffinization of human brain sections, heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0). The following incubation steps were done in a humified chamber and slides were washed between every incubation. Sections were blocked with either 5% milk or 5% donkey serum in tris-buffered saline with 0.5% Tween-20 (TBS-T) (blocking buffer) at room temperature for 1 h. Primary antibodies were diluted in 0.5% blocking buffer and sections were incubated in primary antibody dilution at 4°C overnight. After washing the slides with TBS-T, the appropriate secondary antibodies diluted in 0.5% blocking buffer were applied to the sections at room temperature for 2 h in the dark. For double stainings, tissue sections were washed with TBS-T and incubated with the second primary antibody at room temperature for 3 h. The corresponding secondary antibodies were diluted as mentioned earlier and incubated at room temperature for 2 h. All sections were mounted using Fluoroshield mounting medium containing 4’,6’-diamidino-2-phenylindole (DAPI) (Abcam). Details on the primary and secondary antibodies are listed in Supplementary Table 1. Images were acquired using a Leica DM6 fluorescence microscope equipped with LAS X software at different magnifications as indicated in the figure legends.