Rabbit monoclonal anti α sma antibody

Immunofluorescence staining was performed as previously reported [3 (link)]. Briefly, lung sections were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0, purchased from Bio-Optica, Milan, Italy) for antigen retrieval. Successively, the sections were immune-stained with rabbit monoclonal anti-α-SMA antibody (1:200; Abcam, Cambridge, UK) or rabbit monoclonal anti-NF-kB p65 antibody (1:400, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit Alexa Fluor 568-conjugated IgG (1:300; Invitrogen, San Diego, CA, USA). The negative control was the section in which non-immune rabbit serum was substituted for the primary antibodies. DAPI was used as counterstaining, and representative images were acquired by an Olympus BX63 microscope (Milan, Italy) equipped with Olympus CellSens Dimension Imaging Software version 1.6. The markers’ expression was quantified by densitometric analysis of the intensity of the fluorescence signal in digitized images with ImageJ software. Values are expressed as mean ± SEM of the OD measurements (arbitrary units) of individual mice (20 images each) from the various experimental groups.

After lysis and denaturation of HK2 cells or kidney tissues from each group using the radioimmunoprecipitation assay (RIPA) buffer, proteins (50 μg) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) membranes. After blocking with 1x casein for 1 h to prevent non-specific binding, NC membranes were incubated with the following primary antibodies overnight at 4 °C: (a) rabbit monoclonal E-cadherin antibody (BD Bioscience, USA) diluted 1:100, (b) rabbit monoclonal anti-α-SMA antibody (Abcam, UK) diluted 1:300, (c) mouse monoclonal anti-CTGF antibody (Abcam) diluted 1:200, (d) rabbit polyclonal anti-fibronectin antibody (Proteintech, USA) diluted 1:500, (e) rabbit polyclonal Col3A1 antibody (Proteintech) diluted 1:500, and (f) mouse monoclonal β-actin antibody (Beyotime, China) diluted 1:10,000. The membranes were washed with TBST (Tris-buffered saline with Tween-20, 20 mM of Tris, 140 mM of NaCl, and 0.1% Tween-20) and then probed with 1:1000 diluted secondary antibodies at room temperature (25 °C) for 2 h. Enhanced chemiluminescence (ECL) western blotting kit (APPLYGEN, China) was used to detect the target bands, and β-actin was used as an internal reference to calculate the relative expression levels of proteins in each experimental group.