Anti α sma sc 130617

Human macrophages were seeded in circular coverslips (diameter 18 mm) in 6-well culture plates. Cells were cultured in RMPI medium 0.5% FBS and treated with 1 µM adenosine (ADO; cat# A9251, Merck KGaA, Darmstadt, Germany), 10 ng/mL TGF-β1 (cat# 240-B, R&D systems, Minneapolis, MN, USA), and/or 10 nM MRS1754 every day for one week. Then macrophages were fixed with 3.7% PFA for 10 min and permeabilizated using 1X PBS–0.1% Triton X-100 for 10 min. Cells were blocked using 2.5% normal horse serum and 1% BSA (blocking solution) and then incubated with primary antibody anti-CD68 (ab125212, Abcam, Cambridge, UK), anti-F4/80 (ab111101, Abcam, Cambridge, UK), anti-α-SMA (SC-130617, Santa Cruz Biotechnology, California, USA) and anti-Collagen type I alpha-1 (Col1α1; SC-8784, Santa Cruz Biotechnology, California, USA) in blocking solution overnight at 4 °C. Secondary antibody Alexa 488 and 568 (1:250 dilution; Thermo Fisher Scientific, Massachusetts, USA) were incubated for 45 min and DAPI for 10 min was used as counterstain of nuclei. Finally, samples were washed in 1X PBS and mounted using a fluorescent mounting medium (S3023, Agilent-DAKO, Santa Clara, CA, USA). Images were captured using an epifluorescence microscope (Zeiss) and analyzed with Image J software (NIH, Maryland, USA).

Rat and human kidney tissues were fixed in formalin, embedded in paraffinand 5 μm sections were mounted on silanized slides. Immunodetections were performed as described previously [20 (link)] using the primary polyclonal anti-ENT1 antibody from Protein Tech (II 337-1-A7) and the monoclonal anti-αSMA (sc130617) and anti-A₃AR (sc-13938) from Santa Cruz Biotechnology. The immunosignals were detected using the LSAB+ System–HRP (DakoCytomation).