Immunocytochemistry of Neonatal Rat Cardiomyocytes

For immunocytochemistry (ICC) of NRCs, cells were grown on Lab-Tek II chamber slides (Thermo Scientific, 154461) plated at a density of 1 × 10⁵ cells/well. Subsequently, immunofluorescent microscopy was performed essentially as described previously [48-50 (link)]. Cells were first washed with PBS, fixed for 15 min in 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100 in PBS, and exposed to an antigen retrieval solution (0.1 M glycine, pH 3.5) for 30 min. The fixed cells were incubated in blocking solution (1% BSA, 0.1% cold water fish gelatin, 0.1% Tween-20, 0.05% sodium azide in PBS) for 1 h. Primary antibody incubation was performed overnight at 4°C, whereas the secondary antibody (1:100) along with counterstains were applied for 1 h at room temperature. The following primary antibodies were used: anti-Sigmar1 (1:100, Invitrogen) anti-actin (1:1000, Sigma–Aldrich), anti-TnI (1:1000, Millipore), anti-CHOP (1:100, Cell Signaling, Technologies, Inc.), α-cardiac actin (1:1000, Sigma–Aldrich), anti-XBP1s (1:200, BioLegend). Coverslips were fixed with Vectashield Hardset (Vector Labs, H1400). Nuclei were stained with DAPI (Invitrogen). Cells were imaged using a Leica TCS SP5 Spectral Confocal Microscope loaded with Leica LAS (AF 2.6.3) and NIS-Elements (AR 4.13.04) software.