α sma primary antibody

Hearts were arrested in diastole at 4 weeks, ventricles were weighted and fixed in 10% buffered formalin (see details in the Additional file 1: S9). Cardiomyocyte junctions were identified by immunoperoxidase staining, using a polyclonal rabbit anti-connexin 43 (Cx43) primary antibody (AbCam, Cambridge, UK); arterioles and capillaries were labeled with anti-smooth muscle actin (α-SMA) primary antibody (SantaCruz, Dallas, USA) or anti-PECAM 1 (CD31) primary antibody (SantaCruz, Dallas, USA) respectively. Sections were counter-stained with hematoxylin and examined using light microscopy (Olympus BX43) at 10× to 40× original magnification and digitized by a video system (Olympus DP20 camera) interfaced to a computer with dedicated software (CellSens Dimension, Olympus) for image acquisition and morphometric and/or color analysis. Cx43 was quantified as the percent of myocardial area occupied by positive staining (calculated by averaging the values from ten fields at 10× magnification) for each LV area. The density (number/mm²) of arterioles (10–100 μm in diameter) was counted by averaging values from eight fields randomly chosen for each LV area at 20× magnification. The same was done for capillaries density (<10 μm in diameter), except magnification 40×.

Hs68, PCF54 and PCF55 cells were seeded in DMEM-F12 complete medium at a density of 1 × 10⁴ cells per well in 24-well plates on glass coverslips and grown as a monolayer for 24 h. After that, the cells were rinsed with PBS, fixed, and permeabilized with methanol-acetone (1:1) in -20 °C for 20 min, then washed with PBS and blocked with 2% BSA. Cells were incubated with αSMA primary antibody (sc-32,251, Santa Cruz Biotechnology, USA; 1:50 dilution in PBS, 1% BSA) overnight in + 4 °C and with Cy3-anti mouse secondary antibody (115-165-071, Jackson Immunoresearch, UK) for 1 h in room temperature in dark. Next, cells were washed with PBS and mounted on glass slides in ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, USA) and incubated in + 4˚C, overnight. Fluorescence imaging was done on Leica DM3000 microscope (Leica Microsystems GmbH, Germany).