Primary anti α actinin antibody

At the end of ex vivo experiments, LV were fixated in 4%-PFA and embedded in paraffin. Each LV was cut from apex to base (sections of 4 μM each 150 μM). The paraffin-embedded sections were deparaffinized then rehydrated through an alcohol gradient. Left ventricle sections were incubated with a primary anti α-actinin antibody (1:100, mouse monoclonal; Sigma-Aldrich). Cell nuclei were stained with Hoechst (Life technologies SAS) and endothelial cells with Isolectin B4 (FITC Conjugate; Sigma-Aldrich). After incubation with primary antibodies, sections were washed in PBS, and then incubated (3 h) with secondary antibodies (1:2,000, Jackson ImmunoRes Laboratories, Inc.). Primary and secondary antibodies were diluted in PBS containing 3% BSA and 0.1% Triton X100. Stained sections were mounted in Mowiol (Biovalley). Images were obtained with a Zeiss Axioimager Z3 fluorescent microscope after observation of six different sections of the LV harvested on n = 2 hearts treated by MSC labeled with CM-DiI and analyzed using ImageJ and Adobe Photoshop to prepare the final figures.

For mouse tissue, animals were anesthetized with 2,2,2-tribromoethanol (200 mg kg⁻¹, i.p.; Sigma, St. Louis, MO, USA), and the hearts were quickly removed and perfused transcardially with 4% buffered PFA (pH 7.4). For human cardiac tissues, the preparations were frozen and stored in liquid nitrogen before use. The cardiac tissues were removed, dehydrated and frozen in OCT (opti-mum cutting temperature compound), and 6-μm sections were mounted on glass slides. After blocking, sections were incubated with the primary anti-α-actinin antibody (Cat#A7811, 1:200; Sigma, Saint Louis, USA), anti-Cx43 (Cat#ab11370,1:2000; Abcam, Cambridge, UK), anti-Cx43 (Cat#ab11369, 1:1000; Abcam, Cambridge, UK), or anti-CIP85 antibodies (Cat#sc-86867, 1:100; Santa Cruz Biotechnology, CA) at 4 °C overnight. The cardiac slices were then washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 (1:200) and Alexa Fluor 594 (1:200) (Invitrogen, Camarillo, CA, USA) at room temperature for 1 h. All antibodies were diluted in PBS buffer. The slices were examined under a laser confocal microscope.