Immunohistochemistry and Immunofluorescence Assays

The procedures of these two assays have been described earlier [12 (link)]. Anti‐RYR2 (Proteintech) was diluted 1 : 100 to stain CRC TMA slides. Anti‐phospho‐CREB(Ser133) (Affinity, Changzhou, China) was diluted 1 : 100 in an immunofluorescence (IF) assay. Briefly, for immunohistochemistry (IHC), sections of clinical specimens were deparaffinized with xylene and rehydrated with ethanol, followed by staining with anti‐RYR2 antibody and horseradish peroxidase (HRP)‐linked anti‐rabbit IgG, and further developed with 3,3′‐diaminobenzidine (DAB). TMA sections were further photographed and analyzed by Vecture 2 (PerkinElmer). The same algorithm was used to score every core. For IF, cells on slides were fixed in 4% formaldehyde, followed by staining with indicated primary antibodies and fluorescent secondary antibody (Alexa Fluor 488‐conjugated donkey anti‐rabbit IgG and Alexa Fluor 555‐conjugated goat anti‐rabbit IgG, 1 : 1000) and were photographed with a confocal microscope (Zeiss, Cambridge, UK; LSM 880NLO FILM).