Assessing Tissue Slice Viability

A Dextran uptake assay in combination with ryanodine receptor (RyR) immunofluorescence was performed to assess viability of the tissue slices according to a published method (Pfeuffer et al., 2023 (link)). Briefly, dextran (3 kDa) conjugated to FITC (Thermo Fisher, D3306) at a concentration of 2 mg/mL was incubated for 10 min at 37°C and 5% CO₂ under continuous rocking on the MyoDish culture system platform. The slices were immediately fixed with 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min and then washed three times with PBS for 5 min each. Subsequently, slices were incubated with primary antibody against cardiac RyR (IgG1, mouse, C3-33, Thermo Fisher, Braunschweig, Germany) 1:200 in blocking solution (BS: 5% NGS, 5% BSA, 0.25% Triton-X in PBS) for 4 h at RT or overnight at 4°C, washed three times with PBS for 5 min and incubated with the secondary antibody goat anti-mouse IgG1 AF-555 (Thermo Fisher A-21127) 1:400 in BS for 3 h at RT. After washing, the slices were incubated with wheat germ agglutinin (WGA-AF-647 Thermo Fisher W32466) 40 μg/mL and DAPI (3665, Roth, Karlsruhe, Germany) 1.67 μg/mL in PBS for 3 h. The slices were mounted with Fluoromount G (00-4958–02, Thermo Fisher; F4680 Sigma-Aldrich, Darmstadt, Germany) on a glass microscope slide, covered with a coverslip and equilibrated for 3–7 days at 40%–45% humidity.