Alexa fluor 596 goat anti mouse igg

Five-micrometre cryosections were fixed in ice-cold acetone, permeabilized with 0.3% Triton X-100 and blocked with 10% goat serum. The following primary antibodies were used: rabbit anti-WAVE1 (1:200; Abcam, UK) and mouse anti-synaptopodin (1:100; PROGEN, USA). After three washes, the slides were incubated with Alexa Fluor® 488 goat anti-rabbit IgG (1:200; Invitrogen) and Alexa Fluor® 596 goat anti-mouse IgG (1:200; Invitrogen). Cells on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 10% goat serum. Primary and secondary antibodies as well as Alexa-phalloidin (1:200; Invitrogen) were applied. The slides were mounted with 50% glycerol. Images for each antibody at the same light exposure were obtained by confocal laser-scanning microscopy (TCS-SP5, Leica, Germany). Photographs of WAVE1-stained glomeruli and podocytes were selected randomly and analysed by a person who was blinded to the study groups.

Five-micrometer cryosections were fixed in ice-cold acetone, subsequently permeabilized and blocked with 0.3% Triton X-100 and 10% goat serum. The following primary antibodies were used: rabbit anti-C3, DAF, C3aR (1:25, Santa Cruz, CA, USA), mouse anti-C1q (1:50, Abcam, Cambridge, MA, USA), mouse anti-C3d (1:50, Abrova, Taipei, Taiwan), mouse anti-synaptopodin (1:100, PROGEN, Heidelberg, Germany), rabbit anti-synaptopodin (1:100, Abcam, Cambridge, MA, USA), and rabbit anti-nephrin (1:1000, Sigma, St. Louis, MO, USA). After washing three times, the slides were incubated with Alexa Fluor^®488goat anti-rabbit IgG, Alexa Fluor^®596goat anti-rabbit IgG, Alexa Fluor^®488 goat anti-mouse IgG and Alexa Fluor^®596 goat anti-mouse IgG (1:200, Invitrogen, Carlsbad, CA, USA). The slides were mounted using 15% Mowiol (Sigma, St. Louis, MO, USA). Stained images for each antibody were obtained at the same light exposure using confocal laser-scanning microscopy (Zeiss Lsm510 Meta, Jena, Germany). Images of podocytes stained with each antibody were selected randomly and analyzed by a person who was blind to the study groups.