Immunohistochemical Analysis of Tumor Organoids

Tumour organoids were gently centrifuged at 100 rcf from organoid culture medium and then embedded in 2% agar. Tumour organoids were fixed in 4% paraformaldehyde solution for overnight at room temperature. The samples were replaced with 70% ethanol and later embedded in paraffin blocks. Paraffin sections were cut at 5 μm thickness, attached on slides, heated at 60°C for 1 h and then overnight at 37°C. The slides were stained with hematoxylin and eosin (H&E). IHC staining follows the protocol described^{62 (link)}. Mouse-specific antibodies for CD8α (Cell Signaling Technology, clone D4W2Z, 1:400 dilution), cleaved caspase3 (Cell Signaling Technology, clone D175, 1:400 dilution) and Ki67 (Cell Signaling Technology, clone D3B5, 1:400 dilution) were used for IHC staining. The DAB peroxidase substrate kit (Vector laboratories Inc.) was used to visualize the antibodies. For IF analysis, the sample slides were blocked 3% BSA for 40 min after hydration. The slides were stained with primary antibodies including EpCAM (Abcam, 1:400 dilution), FITC-conjugated anti-α-SMA (Genetex, 1:400 dilution) and AF594-conjugated anti-CD31 (Biolegend) for 1 h. For EpCAM staining, the slide was further stained with secondary antibody Alexa Fluor 647-onjugated anti-rabbit IgG (Biolegend, 1:200) for 40 min. The fluorescence images were captured under a Leica DM4B microscope.