Anti cardiac troponin t ctnt

Samples were fixed with 4% PFA (Sigma) at room temperature for 30 min, washed three times with DPBS, permeated with 0.2% v v⁻¹ Triton X-100 (Sigma Aldrich) in DPBS for 15 min, washed with DPBS and incubated with 10% (v v⁻¹) donkey serum (VWR International), 5% (w v⁻¹) bovine serum albumin (Sigma Aldrich), and 0.3 M glycine (Sigma Aldrich) for 1 h. A combination of two antibodies and dilutions from the following were chosen to characterize the morphology of single isolated hiPSC-CMs: anti-α-actinin, 1:200 (EA-53, monoclonal, Sigma); anti-cardiac troponin T (cTnT), 1:200 (Abcam); anti-Connexin-40, 1:200 (Abcam); anti-Connexin-43, 1:100 (Abcam); anti-Connexin-45, 1:500 (Abcam); anti-MYL2, 1:140 (Abcam); and anti-MYL7 1:400 (Abcam) overnight at 4 °C in DPBS. Afterward they were washed extensively with DPBS before incubating at a 1:400 dilution with two appropriate secondary antibodies(donkey anti-mouse immunoglobulin G, donkey anti-rabbit or donkey anti-goat (all IgG, H + L, Thermo Fisher Scientific)) in DPBS for 1 h. Nuclei were stained with 1:500 DAPI (Thermo Fisher Scientific) in DPBS for 1 min. The samples were mounted with Fluoromount Aqueous Mounting Medium (Sigma Aldrich) and imaged with a Zeiss LSM-780 confocal microscope. Cx-43 surface area analysis were performed using Fiji image analysis software (N ⩾ 5, n ⩾ 3).

For flow cytometry and imaging flow cytometry, cells were treated with the FIX and PERM^® Kit (Thermo Fischer Scientific, Waltham, MA, USA) and incubated for 1 h at RT with anti-α-myosin heavy chain (α-MHC; 1:100; Abcam, Cambridge, UK), anti-cardiac troponin T (cTnT, 1:100; Abcam, Cambridge, UK), anti-α-sarcomeric actin (α-SA; 1:100; Abcam, Cambridge, UK), anti-sarcolemmal Ca²⁺ channel (CACNA1C, 1:100; Abcam, Cambridge, UK), and anti-sarcoendoplasmic reticulum Ca²⁺ ATPase protein (Serca2, 1:100; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), followed by incubation with the appropriate secondary antibody conjugated with Alexa Fluor 488, PE-Alexa Fluor 647, or PE-Cy7 (1:200; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [53 (link)]. Cells incubated with isotypes (all from BD) were used as negative controls [54 (link)].
Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) or CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).