Lsm5 pascal

The kidney tissues were dissected from the mouse, fixed in OCT compound, and stored at −80°C until use. Several 6 μm thick frozen sections were prepared and fixed with ice-cold methanol, air-dried for 30 min at room temperature, and then blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.05% Tween-20 in PBS. After washing with Tween in PBS (PBST; 0.1% Tween-20 in PBS), the slides were coincubated with VPAC1 antibody (1 : 50 dilution, Santa Cruz: sc-30019), podocalyxin (1 : 200 dilution, R&D systems: MAB1556), and toll-like receptor 4 (1 : 50 dilution, Santa Cruz: sc-10741). The slides were imaged by the Axio Observer D1 (ZEISS) or with a confocal laser scanning microscope (Leica LSM5 PASCAL). The images were processed using Adobe Photoshop.

The kidney tissues were dissected from the mouse, fixed in O.C.T. compound, and stored at −80°C until use. Several 4–8 μm frozen sections were prepared and fixed with 4% paraformaldehyde or ice-cold methanol and then blocked with blocking buffer, containing 2% bovine serum albumin (BSA), and 0.05% Tween-20 in PBS. After washing with Tween in PBS (PBST; 0.1% Tween-20 in PBS), the slides were co-incubated with nephrin antibody (1:500 dilution), PECAM antibody (BD Pharmingen #550274, 1:200 dilution), WT-1 antibody (Santa Cruz, sc-129, 1:50 dilution), Neuropilin-1 antibody (kindly provided by Dr. Fumikazu Sudo, National Institute of Neuroscience, Japan, 1:200 dilution) and Hoechst for nuclear staining. The slides were imaged by the Axio Observer D1 (ZEISS) or with a confocal laser-scanning microscope (Leica LSM5 PASCAL). The images were processed using Adobe Photoshop.