Immunofluorescence Assay for CASP9

The tissues were sectioned and embedded in paraffin. Sections were incubated overnight at 4°C with anti-CASP9 antibody (1:100 dilution; proteintech, China). Slides were washed with phosphate-buffered saline (PBS) and incubated with a goat anti-rabbit IgG secondary antibody conjugated with fluorescein isothiocyanate (ZSDB-BIO, China) for 30 min with washed slides. After washing with PBS, they were incubated with an antifade reagent (Invitrogen, United States). Staining was visualized to determine protein expression levels using an Olympus CX41 fluorescence microscope (Olympus, Japan). The analysis results were performed using Image J software.