Mouse anti α smooth muscle actin α sma clone 1a4

Cells grown on coverslips were fixed with 3% paraformaldehyde and stained with primary and fluorescently labeled secondary antibodies. The following antibodies were used: mouse anti-α-smooth muscle actin (α-SMA) (clone 1A4, Sigma, Darmstadt, Germany), mouse anti-synaptopodin (clone D-9, Santa Cruz, Santa Cruz, CA, USA), mouse anti-cytokeratin 18 (CK18) (clone RGE-53, Merck Millipore, Darmstadt, Germany), rabbit anti-fibronectin (Sigma), mouse anti-integrin α_vβ₃ (clone LM609, Millipore), mouse anti-CD31 (Dako, Glostrup, Denmark), and mouse anti-N protein PUUV (A1C5, Progen, Heidelberg, Germany). Cell nuclei were stained by Hoechst 33342 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using an Axiocam 506 mono camera attached to an Axio Observer.D1 inverted microscope (Carl Zeiss, Oberkochen, Germany). For Western blot analysis, the following primary antibodies were used: rabbit anti-PUUV N protein and mouse anti-α-tubulin (Sigma). Loading was verified by the detection of tubulin on the same membrane. Detection was performed by using near infrared fluorescent dye (IRDye)-conjugated secondary antibodies and an Odyssey CLx infrared imaging system (Li-Cor, Lincoln, NE, USA).

Immunoblot of the renal cortex was carried out as described previously [13 (link)]. Monoclonal antibodies used in this study were: mouse anti-α-smooth muscle actin (α-SMA) clone 1 A4, used at a dilution of 1:1000, from Sigma-Aldrich, St. Louis, MO, USA; mouse anti-vimentin (used at 1:1000) from Dako Inc., Santa Clara, CA, USA; mouse anti-TRPC5 (used at 1:100) from NeuroMAB, Davis, CA, USA; mouse anti-pro-interleukin−1β (used at 1:1000) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Polyclonal anti-rabbit NLRP3 (used at 1:1000) was from Abcam (Cambridge, MA, USA; anti-TRPC6 (used at 1:1000), and anti-TRPC3 (used at 1:1000) was from Alomone Laboratories (Jerusalem, Israel). All experiments were quantified by densitometry using Image J™ software. Type I procollagen levels in serum were determined using a commercial assay (LSBio, Inc., Seattle, WA, USA) according to the manufacturer’s instructions. β2–microglobulin (β2–MG) levels in the urine were assessed using an assay from ICL Lab (Portland OR, USA). Albumin was measured in 24-h urine samples using a kit from Ethos Biosciences (Philadelphia, PA, USA).