Fluoview fv3000 laser scanning microscope

For the microscopy-based assay, SK-BR-3 and MDA-MB-468 tumour cells were seeded in chamber slides (LabTEK) and grown to approximately 50% confluence. Cells were then incubated with HER/SLAMF7–conjugated NPs (BiTN_HER) (HER:SLAMF7 ratio=1:1) at 37°C for 1 h, with non-targeting IgG/SLAMF7-NPs (IgG-SNP) (IgG:SLAMF7 ratio=1:1) used as a control. The cells were then washed three times with PBS to remove unbound NPs and fixed with 1% paraformaldehyde for 15 min. Cells were then blocked with 5% BSA and stained with allophycocyanin-conjugated anti-human SLAMF7 (clone 162.1, BioLegend, #311809, 1:100) and AlexaFluor 488-conjugated anti-human IgG antibody (Invitrogen, #A11013, 1:100) overnight. Cell nuclei were counterstained with DAPI and the samples were mounted with an antifade mounting medium (ThermoFisher) before imaging. Images were obtained with Olympus Fluoview FV3000 laser scanning microscope with a ×40 oil emersion objective, numerical aperture (NA) 1.4.

Tumour tissues were carefully isolated and fixed in 4% buffered formalin phosphate solution (Fischer Scientific) for 24 h, followed by 70% ethanol solution overnight. The tissues were embedded in paraffin, and 5-μm tissue slices were prepared. For the immunostaining studies, tumour slides were pre-blocked with a blocking solution containing 5% BSA and 10% control normal serum and then stained with primary antibodies including CD4 (clone 4SM95, eBioscience, #14-9766-82, 1:50), CD8 (clone 4SM15, eBioscience, #100702, 1:50), F4/80 (clone CI:A3-1, Abcam, #6640, 1:100), Iba-1 (pAb, Abcam, #ab5076, 1:1000), CD11c (clone N418, eBioscience, #14-0114-82, 1:100), pIRF3 S396 (clone D6O1M, Cell Signaling, #29047, 1:50), PD1 (clone D7D5W, Cell Signaling, #84651, 1:200), and PDL1 (clone D5V3B, Cell Signaling, #64988, 1:200) at 4 °C overnight. After washing, the appropriate fluorescence-labelled secondary antibodies (eBioscience) were added for 2 h at room temperature in the dark. Cell nuclei were counterstained with DAPI and the samples were mounted with an antifade mounting medium (ThermoFisher) before imaging. Fluorescence images were obtained with an Olympus Fluoview FV3000 laser scanning microscope and were further analysed with Image J software.