Rabbit polyclonal anti tgf β

Mouse anti-β-actin monoclonal antibody was obtained from Sigma (St. Louis, MO, USA). Rabbit polyclonal anti-TGF-β, rabbit polyclonal anti-α-smooth muscle actin (SMA), horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG), rabbit polyclonal anti-type IV collagen, rabbit polyclonal anti-TGF-β, rabbit anti-Smad2/3 antibody, rabbit anti-pSmad2/3 antibody and horseradish peroxidase-labeled goat anti-rabbit IgG were purchased from Abcam (Cambridge, MA, USA). The spare kidney tissue was removed and thawed and homogenized for radio immunoprecipitation. The homogenate was centrifuged for 10 minutes at 12 000 rpm in a 4°C cryogenic high-speed centrifuge, and then, the supernatant was collected. Quantitative 70 μg protein samples were prepared by gel electrophoresis and then transferred to a cellulose acetate membrane. A 1x casein solution was used to block the membranes for 1 hour after transfer. After blocking, the membrane was transferred to the refrigerator and stored at 4°C and incubated overnight with the first antibody. Then, the samples were washed thoroughly and incubated with the second antibody. Images of protein imprinting were analyzed using Image J 1.42 software after antibody incubation.

Mice under deep anesthesia were transcardially perfused with ice-cold PBS, followed by immersion in 4% paraformaldehyde. Coronal sections were prepared, blocked and then cut. Frozen sections were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-PDGFR-β (1:50, Cell Signaling Technology), mouse monoclonal anti-NG2 (a marker of cerebrovascular pericyte 1:100, Santa Cruz), rabbit polyclonal anti-TGF-β (1:100, Abcam), rabbit polyclonal anti-Ki-67 (1:100, Cell Signaling Technology), rabbit polyclonal anti-MCP-1 (1:200, Abcam) and rabbit polyclonal anti-TNF-α (1:50, Abcam). Then, the sections were incubated with appropriate host secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 637 (1:200, Invitrogen, USA). All sections were stained with 6-diamidino-2-phenylindole (DAPI, CA, USA) and photographed with a confocal microscope (TCS SP5, Leica).

Total protein was extracted from kidney tissues with RIPA lysis buffer containing protease inhibitors. The protein concentration was assayed using a BCA kit. Seventy micrograms of protein were electrophoretically separated on 10% SDS-PAGE gels and then transferred to a cellulose acetate membrane. After blocking for 1 h with a 1× casein solution, the primary antibody was added and incubated overnight at 4 °C. The mouse monoclonal anti-β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Rabbit polyclonal anti-TGF-β, rabbit polyclonal anti-α-smooth muscle actin (α-SMA), and rabbit polyclonal anti-type I collagen antibodies were purchased from Abcam (Cambridge, MA, USA). After washing, the membranes were incubated with the corresponding secondary antibody. Images were analyzed using ImageJ 1.42 software.