Mouse anti rat tgf β1 monoclonal antibody

Specimens were deparaffinized and rehydrated with xylene and 80, 90, 95 and 100% ethanol. Following washing three times with PBS, sections were placed in 0.01 mol/l citrate buffer (pH 6.0), heated in a microwave (92–95°C, 15 min) for antigen retrieval and cooled to room temperature. Following antigen retrieval, primary antibodies, including mouse anti-rat α-smooth muscle actin (α-SMA) monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab7817) and mouse anti rat TGF-β₁ monoclonal antibody (Abcam; cat. no. ab64715) were used at dilutions of 1:75 and 1:50, respectively, and incubated overnight at 4°C. Immunoglobulin G-conjugated horseradish peroxidase (HRP) goat anti-mouse polyclonal antibody (1:500; cat. no. ZB-2305; Beijing ZSGB Biotech. Co., Ltd., Beijing, China) and 3,3-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) were employed to visualize antibody binding (control time under a microscope at room temperature). Positive staining was defined as the presence of tan-yellow color granular staining. Optical density values of positive areas in each slice were calculated using Image-Pro Plus 6.0 analysis software (Media Cybernetics, Inc., Rockville, MD, USA).

Kidney tissues were resuspended in ice-cold radioimmunoprecipitation assay buffer (Bio-Rad Laboratories, Inc.) for lysis. For the protein assay, a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China) was used. Approximately 30 µg protein samples were loaded, separated on 9.0% Tris-Glycine-SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were then incubated for 1 h in a blocking solution containing 5% non-fat milk in a PBS 0.05% Tween solution. Following blocking, membranes were incubated with the mouse anti-rat α-SMA monoclonal antibody (1:250) or the mouse anti rat TGF-β₁ monoclonal antibody (1:500; Abcam) overnight at 4°C. Membranes were subsequently incubated with polyclonal goat anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at 37°C. Immunoreactive bands were detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Band intensities were quantified using Image J 1.32 software (National Institutes of Health, Bethesda, MD, USA). β-actin was used as an internal loading control.