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.
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.
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