Immunostaining of Frozen Tissue Sections

The frozen tissue sections were fixed in −20°C acetone for 10 min and washed in phosphate-buffered saline (PBS). Background was blocked with 2% bovine serum albumin, 0.3% Triton-X100, and 5% goat serum (Invitrogen, Carlsbad, CA) in PBS for 30 min at room temperature (RT). Three primary antibodies were combined in each protocol, diluted according to Supplementary Table S2. Primary antibodies were incubated at 4°C overnight. The sections were washed, and the following secondary antibodies were added for 1 h at RT: goat anti-mouse Alexa Fluor 546, goat anti-rabbit Alexa Fluor 546, or goat anti-rabbit Alexa Fluor 647 (Invitrogen). To enable the use of two mouse primary antibodies, the cTnT antibody was conjugated with Alexa 488 fluorochrome using Zenon Kit (Invitrogen). The samples were fixed using Histofix (Histolab, Gothenburg) for 15 min, washed, and mounted with prolong gold antifade reagent with nuclei staining 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Corresponding isotype controls for primary antibodies and did not show specific staining.