Anti αsma antibody a2547

TAA was purchased from Sigma-Aldrich (St. Louis, MO, USA), and recombinant human transforming growth factor beta (TGF-β), 5 ng/mL was purchased from R&D Systems (Minneapolis, MN, USA). The anti-clusterin (sc-6420) and anti-phospho-Smad2/3 (sc-11769) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-collagen antibody (ab-34710) was purchased from Abcam (Cambridge, UK), and the anti-αSMA antibody (A2547) was purchased from Sigma-Aldrich. The anti-GAPDH (cs-2118), anti-phospho-Smad3 (Ser423/425), and anti-Smad3 (cs-9520) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Immunohistochemistry was performed as previously described [29 (link)]. We stained 4 μm thick sections from kidneys harvested at day 21, with mouse monoclonal anti-α-SMA antibody (A-2547; Sigma-Aldrich, Saint Louis, MO, USA) and rabbit polyclonal anti-CD3 antibody (IR503; Dako, Santa Clara, CA, USA). Reaction products were detected using the avidin-biotin-peroxidase complex method (Vector Laboratories, Burlingame, CA, USA), and color reactions were developed in 3,3-diaminobenzidine (Sigma-Aldrich, Saint Louis, MO, USA) and hydrogen peroxide. Five non-overlapping fields of the renal cortex were selected (×100, n = 5), and images were qualitatively analyzed using ImageJ software.