Application suite las v5

For immunohistochemical studies, hearts were snap frozen in liquid nitrogen, or frozen in isopentane at −50°C, or incubated overnight in 4% PFA followed by overnight incubations in 20% and 30% sucrose in PBS, prior to embedding in OCT and storage at −80°C. 10–15 µm sections were cut using a cryostat (Bright instruments), air dried and immersed in 4% PFA in PBS or in acetone at −20°C for 15 min and washed for 3×5 min in 0.1% PBS-Triton X-100. Blocking was achieved by incubation with 5% BSA-C (Aurion) in 0.1% PBS-Triton X-100 for at least 30 min at RT. Immunolabeling with primary antibodies was performed in 0.1% PBS-Triton X-100, 1% BSA-C overnight in a humidity box at 4°C as described previously [15] (link). Sections were washed 3× in PBS, incubated for 60 min at RT in a dark box with the anti-rabbit (FITC Invitrogen 1∶1000 in PBS), washed 3× in PBS and counterstained with DAPI (Invitrogen). Sections were mounted in Vectashield mounting medium (Vector Laboratories). Sections were examined using the Leica TCS SP4 laser scanning confocal microscope and analysed with Leica Application Suite (LAS) v5 (Leica Microsystems, Heidelberg, Germany).

For immunohistochemistry, hearts were snap frozen in liquid nitrogen, prior to embedding in OCT. 6–10 μm sections were cut using a cryostat (Leica), air dried and fixed in 4% PFA, (paraformaldehyde) in PBS or in acetone, at -20^°C for 15 min, followed by washing in 0.1% PBS-Triton X-100 [14 (link)]. Blocking was achieved by incubation with 5% BSA-C (Aurion) in 0.1% PBS-Triton X-100, for at least 30 min at RT. Immunolabelling with primary antibodies (anti-collagen VI 1 in 100 (600-401-108-05, Rockland); anti-vinculin 1 in 200 (V4505, Sigma) was performed in 0.1% PBS-Triton X-100, 1% BSA-C, overnight in a humidified box at 4^°C. Sections were washed 3 times in PBS, incubated for 60 min at RT in a dark box, with an anti-rabbit secondary antibody (FITC Invitrogen, 1:1000 in PBS), washed 3 times in PBS and counterstained with phalloidin (Sigma) and DAPI or draq5 (Invitrogen). Sections were mounted in Vectashield mounting medium (Vector Laboratories). Sections were examined using the Leica TCS SP4 laser scanning confocal microscope and analysed with Leica Application Suite (LAS) v5 (Leica Microsystems, Heidelberg, Germany).