Immunohistochemistry for activated caspase-3

Apoptosis in the center and outer layer of each tumor nodule (sampled from the greater omentum) was evaluated by means of IHC staining for activated caspase-3. Ten μm frozen sections were stored at -80°C until day of analysis. Sections were washed for 5 minutes with aqua destillata followed by cell permeabilization for 5 minutes using PBS-0.03% Triton X-100 (Fluka, Sigma-Aldrich Chemie, Steinheim, Germany). Non-specific interactions were minimized through blocking by incubation in 0.3% hydrogen peroxide (Sigma-Aldrich Chemie, Steinheim, Germany) in methanol (VWR Chemicals, Fontenay-sous-Bois, France) for 20 minutes, and subsequently incubated with 20% pre-immune goat serum for 45 minutes. Thereafter, sections were incubated overnight with the primary antibody rabbit active caspase-3 (1/20 dilution, 3015-100, Gentaur, Kampenhout, Belgium). Next, sections were incubated with the secondary antibody, goat anti-rabbit biotin (E043201-8, Agilent Technologies, Diegem, Belgium) for 45 minutes followed by a 30-minute incubation with Streptavidin-HRP (1/100 dilution, Dako, Glostrup, Denmark) and 8 minutes incubation with the TSA™ Cyanine 3 kit (NEL704A001KT, Perkin-Elmer, Nossegem, Belgium). Cell nucleus was stained by means of DAPI staining (1/1000 dilution in PBS) for 10 minutes. Every step was interspersed with 3 times 5-minute wash steps in PBS. For negative controls, the primary antibody was omitted. Microscopy was performed using a ELYRA PS.1 epi-fluorescence microscope (Carl Zeiss, Jena, Germany). Wide-field images of whole tumor cross sections were obtained by means of tile scans using a Plan-Apochromat 20x/NA0.8 Ph2 objective. DAPI and Cy3 were sequentially excited with lasers at 405 nm and 561 nm respectively. The emission light of DAPI was collected at 420 – 480 nm and the Cy3 emission light was collected at 570 – 620 nm. The acquired image resolution for each tile was 1280×1280 pixels² at a pixel size of 203 nm. For regions with very high cell density, additional images were acquired with structured illumination microscopy using a Plan-Apochromat 63x/NA1.40 Oil DIC (Carl Zeiss, Jena, Germany) objective utilizing the same optical filters as described above, resulting in images with 1280×1280 pixels² at a pixel size of 64 nm. For each structured illumination image, 25 raw images (combination of 5 angles and 5 translations of the diffraction grating) were recorded and subsequently reconstructed into a final high resolution image using automatic 2D processing in ZEN software (version 2011, Carl Zeiss, Jena, Germany). Cells positive for activated caspase-3 were counted manually in two sections for each tumor nodule (center and outer layer) and expressed as number of cells/mm².