2.10. Western blot analysis of markers of fibrosis

Kidneys were processed for western blot analysis as previously described [14]. In brief, 30–50 mg total protein samples were denatured and loaded onto 10 % Bis-Tris gels (Invitrogen). After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were blocked for 1 h with blocking buffer (5 % nonfat milk in PBS), then incubated overnight at 4˚C with the primary Abs against type I collagen and α-smooth muscle actin (α-SMA) diluted in blocking buffer with 0.1 % Tween 20. Mouse anti-GAPDH Ab (Santa Cruz Biotechnology) was used as a loading control. After incubation, the membranes were washed and incubated for 1 h with IR Dye 800 goat anti-rabbit and IR Dye 680 goat anti-mouse (LI-COR Biosciences) diluted in blocking buffer plus 0.1 % Tween 20. The blots were then washed, and proteins were visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences). We considered unreliable measurements of markers of fibrosis by Western Blot analysis in kidneys from mice that were found dead, because proteases start to activate soon after the death. Therefore, kidney from mice that were found dead were excluded from this analysis.