Primary antibody for cleaved caspase 3

Following PET imaging, tumour tissues were collected, fixed in formalin, embedded in paraffin, and sectioned. Immunohistochemistry for cleaved caspase-3 was performed on tumour sections to evaluate apoptosis. Briefly, after antigen retrieval, sections were incubated with a primary antibody for cleaved caspase-3 (Cell Signaling, 1:500) overnight at 4 °C, and visualized using biotin-conjugated secondary antibodies (Vectastain ABC Kit; Vector Labs) and 3, 3′-diaminobenzidine (DAB; Dako). Sections were then counterstained with haematoxylin and mounted with DPX (Sigma Aldrich).
Image acquisition of tumour sections was performed using the AxioScan digital slide scanner (Zeiss), and QuPath (Queen’s University Belfast, Northern Ireland) image analysis software was used for IHC quantification [35 (link)]. Briefly, regions of interest were defined to include viable tumour tissue, excluding necrotic areas. Cell-based analysis was performed with automated cell segmentation based on colour: haematoxylin in the blue and DAB in the red channel. To avoid artefacts, a threshold for minimum cell area and variance of haematoxylin staining was set. Positive cells were selected based on mean and maximum intensity of DAB. Data were generated by calculating the percentage of the DAB-reactive cells in relation to the total number of cells in the regions of interest.

Colo320 cells were seeded in 24-well plates and were infected the next day when cells reached a confluence of about 80% with PD-H and PD-SK at MOI 0.1. Twenty-four h after infection cells were fixed with 4% formaldehyde (Carl Roth) for 15 min, washed three times with PBS and incubated with 500 µl blocking solution (PBS, 5% goat serum, 0.3% Triton X-100) for 1 h. Blocking solution was removed, and cells were incubated with primary antibody for cleaved caspase-3 (Cell Signaling Technology) at 4 °C overnight. After washing with PBS, incubation with secondary antibody Goat anti-Rabbit IgG (H + L) Alexa Fluor™ 488 (Thermo Fisher Scientific) was carried out for 2 h. Thereafter, cells were washed three times with PBS followed by co-staining with 1 µg/mL of 40,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min. Cells were washed twice again with PBS and images were taken by fluorescence microscopy (Observer Z1, Carl Zeiss, Oberkochen Germany).