Mouse anti α actinin

XMU‐MP‐1 (Fan et al., 2016) was obtained from MedChem Express. Phenylephrine was obtained from Sigma (P6126). Antibodies used in this study include mouse anti‐α‐actinin (Sigma, cat no A7811), rabbit anti‐phospho‐MOB1 (Cell Signalling Technology, #8699S), rabbit anti‐MOB1 (Cell Signalling Technology, #13730S), rabbit anti‐active‐YAP (Cell Signalling Technology, #29495S), anti‐β‐actin HRP conjugate (Cell Signalling Technology, #122625), and anti‐Ki67 (Abcam, ab15580). Secondary antibodies used are Alexa Fluor® 647 anti‐mouse IgG (Jackson ImmunoReasearch Laboratories, Inc., 115‐605‐003), Alexa Fluor® 488 anti‐rabbit IgG (Jackson ImmunoReasearch Laboratories, Inc., 111‐545‐144), and anti‐rabbit IgG (Cell Signalling Technology, #7074S).

At room temperature, cardiac tissues were fixed inside the chips with 4% paraformaldehyde for 1 ​h. For immunostaining, tissues were embedded at optimal cutting temperature and cryo-sectioned to 20 ​μm thickness. After permeabilization (0.2% Triton X-100), 3% bovine serum albumin (BSA) was used to block the samples. The following primary antibodies were used for immunostaining: mouse anti-α-actinin (Sarcomeric) (Sigma-Aldrich SAB4200813; 1:200); mouse anti-cTnI (Sigma-Aldrich MFCD01864286; 1:200); rabbit anti-connexin-43 (Cell Signaling 3512 ​S; 1:75); and the proper secondary antibodies: Alexa Fluor 594 goat anti-mouse IgG (Invitrogen A-21044, 1:200); Alexa Fluor 488 goat anti-mouse IgG (Invitrogen A-10680, 1:200); Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen A-11037, 1:200). The nucleus was stained with DAPI (Beyotime, 1:200). A Revolution XD confocal laser scanning microscope (Andor) was used for imaging.

FDB fibers dissociated from control mice and mice < 1 hr after exercise were plated on glass bottom dishes and fixed in 4% paraformaldehyde for 20 min at room temperature. Following blocking for 1 hr at room temperature in 10% BSA in 1X PBS-T (PBS + 0.1% Triton X-100), fibers were co-labeled overnight at 4°C using primary antibodies for GFP (Rabbit polyclonal antibody, Thermo Fisher Scientific, Cat. # A11122, 1:3000) and either α-actinin (Mouse anti α-actinin, Sigma, Cat # A7811, 1:750) or the type1 ryanodine receptor (RYR1- Mouse anti RYR1 Cat # 34C, DHSB University of Iowa, 1:30). All primary antibody incubations were in 2% BSA in 1X PBS-T. Following three 10 min washes in PBS-T, samples were incubated with a 1:500 dilution of Goat anti Rabbit Alexa fluor 488 (Molecular Probes Cat # A11034) and Goat Anti Mouse Alexa fluor 594 (Molecular Probes Cat # R37121) for 1 hr at room temperature. Following three 10 min washes in 1X PBS-T, samples were imaged using an Olympus FV1000 laser scanning confocal microscope (Olympus Scientific Solutions, Wlatham, MA) and a 100X, UPlanSAPO NA 1.4 oil immersion objective. Alexa 488 and 594 were sequentially excited at 488 and 559 nm and detected at 515 and 617 nm respectively.

Standard histological procedures were used (Roux et al., 2015 (link)). Heart from Hoxb1^GoF;Mef2c-Cre and littermate controls were fixed in neutral-buffered 4% paraformaldehyde in PBS, rinsed, dehydrated, paraffin-embedded and tissue sections cut at 8 μm. Sections were stained with Harris’ hematoxylin and eosin (H and E) (Sigma). For immunostaining embryos from Hoxb1^-/- or Hoxb1^GoF;Mef2c-Cre and littermate controls were fixed at 4°C for 20 min in 4% paraformaldehyde, rinsed in PBS, equilibrated to 15% sucrose and embedded in O.C.T. Cryo-sections were cut at 12 μm, washed in PBS and pre-incubated in blocking solution (1%BSA, 1% Serum, 0.2% Tween20 in PBS). Primary antibodies were applied overnight at 4°C, followed by secondary detection using Alexa Fluor conjugated (Molecular Probes) secondary antibodies. Sections were photographed using an AxioImager Z2 microscope (Zeiss) and photographed with an Axiocam digital camera (Zen 2011, Zeiss).
The following primary antibodies were used in this study: rabbit anti-Hoxb1 (Covance; 1/200), rabbit anti-GFP (Life Technologies; 1/500), mouse anti-αactinin (sigma; 1/500), mouse anti-MF-20 (DHSB; 1/100), Rabbit anti-Caspase3 (Cell Signaling Technology, 1/300), rabbit anti-phospho-Histone H3 (Millipore; 1/400), and mouse anti-Islet1 (DSHB; 1/100), rabbit anti-Tbx1 (Isbio Ls-C31179, 1/100), goat anti-Tbx5 (Santa Cruz sc-7866, 1/250).