M0851 mouse monoclonal antibody

Protein expression of the fibrotic and inflammatory markers was studied by Western blot. Following measurement of maximum contractility, LMS were quickly washed with PBS and the rings were dissected off with a razor blade. The LMS was then immediately snap‐frozen in liquid nitrogen and stored at −80°C for analysis. Total protein samples were prepared as previously described.¹⁷ LMS were crushed in a fine powder (20 mg) using a pestle and mortar under liquid nitrogen, suspended in 0.1 mg/μL SB20 solution and sonicated for approximately 1 min. After the addition of 2‐mercaptoethanol (2.5% final concentration), proteins (50 μg) were fractionated on 8% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), electrophoretically transferred to 0.2 μM polyvinylidene difluoride (PVDF) immunoblot membranes (BIO‐RAD) and probed for Gapdh (PA1‐987 rabbit polyclonal antibody, Invitrogen, 1:2000), αSMA (M0851 mouse monoclonal antibody, Dako, 1:1000), Collagen I (ab34710 rabbit polyclonal antibody, Abcam, 1:1000) and CD45 (ab10558 rabbit polyclonal, Abcam, 1:500) overnight at 4°C. Bands of interest were detected using Alexa Fluor 488 donkey anti‐mouse IgG and Alexa Fluor 546 anti‐rabbit IgG (1:2000), following an incubation time of 3 h at room temperature. Blots were scanned and quantitative analysis was performed using ImageJ software.

Following culture, LMS were washed in PBS and then fixed in 4% formaldehyde solution for 15 min at room temperature. LMS were permeabilized in 1% Triton X‐100 and blocked in 10% foetal bovine serum, 5% bovine serum albumin and 10% horse serum in PBS solution for 3 h at room temperature. LMS were incubated with the corresponding primary antibody αSMA (M0851 mouse monoclonal antibody, Dako, 1:1000), Collagen I (ab34710 rabbit polyclonal antibody, Abcam, 1:1000), Vimentin (pa1‐10003 chicken, Invitrogen, 1:4000), Cardiac troponin (ma5‐12960, Thermofisher, 1:1000), YAP1 (sc‐101199, Santa Cruz, 1:100) in PBS at 4°C overnight, and then washed three times for 30 min. LMS were incubated in the secondary antibody in PBS for 2 h at room temperature. LMS were then washed three times for 30 min, stained with Hoechst (Thermofisher, 1:1000) during 15 min, washed again three times for 15 min and stored in PBS at 4°C. Immunolabelled slices were imaged using a confocal microscope (Zeiss LSM‐870).