Anti α actinin

Cardiomyocytes were fixed with 4% paraformaldehyde. After permeabilizing the cells using Triton X-100 and blocking the active sites in blocking solution (0.2% Triton X-100, 5% normal serum and 1% bovine serum albumin in phosphate buffered saline) for 30 min at room temperature, primary antibodies, including anti-α-actinin (Abcam), anti-connexin-43 (Santa Cruz), anti-MyHC (Abcam), anti-plakoglobin, and anti-desmin (Novus Biologicals) were applied. After being thoroughly washed, secondary antibodies (goat-anti-mouse and goat-anti-rabbit) conjugated with FITC or Alexa-568 were applied. The labeled cells were then immersed in mounting solution containing DAPI and imaged under a fluorescence microscope. Sarcomeric structure was observed using our lab-built TPEF-SHG imaging system using a procedure similar to that in our previous report [Liu et al., 2013a (link)]. Briefly, cells were excited by an 830-nm femtosecond laser beam, and the two-photon fluorescence signal emitted by the fluorescently labeled proteins was collected from the TPEF channel while the SHG signal from the myosin filaments was collected from the SHG channel. The TPEF and SHG signals were collected simultaneously and 2D images were processed and reconstructed using ImageJ software (http://rsbweb.nih.gov/ij/) and edited in MicrosoftV® Paint.

Cells were fixed with 4% PFA in 1X DPBS for 10 minutes, washed twice 1X PBS, permeabilized for 20 minutes in 0.1% Triton X-100 1X PBS, washed twice with 1X PBS, blocked for at least 60 minutes in 5% BSA 1X PBS, stained overnight at 4°C with primary antibody diluted in 5% BSA 1X PBS, washed three times with 1X PBS, stained for 60 minutes with secondary antibody diluted in 5% BSA 1X PBS, and lastly washed three times with 1X PBS before imaging. Primary antibodies and relevant dilutions were as follows: anti-Tuj1, 1:500 (Abcam, # ab7751); anti-Alb, 1:500 (Abcam, ab207327); anti-cTnT, 1:400 (Thermo Fisher, # MA5-12960); anti-αActinin, 1:500 (Abcam, ab68167); anti-Connexin-43, 1:500 (Sigma, C6219). Secondary antibodies and relevant dilutions were as follows: AlexaFluor 488 anti-mouse, 1:500 (Jackson Immuno, # 715-545-150), AlexaFluor 647 anti-mouse, 1:500 (Jackson Immuno, # 715-605-150), AlexaFluor 488 anti-rabbit, 1:500 (Jackson Immuno, # 715-545-152), AlexaFluor 647 anti-rabbit, 1:500 (Jackson Immuno, # 715-605-152). The quantification of the images was performed with CellProfiler(Stirling et al., 2021 (link)).