Immunofluorescence Staining of Kidney Tissue

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.