Aqueous mounting medium

30,000 SK-MEL-28 melanoma cells were seeded on coverslip placed into 24-well culture plate and then incubated for 24 h in DMEM supplemented with 10% FBS at 37°C in 5% CO₂. Cells were fixed with 4% paraformaldehyde in PBS (w/v) and incubated for 10 min at room temperature. After washing with PBS, cells were then incubated with biotinylated NC1(XIX), NC1(XIX) and/or anti-αvβ3 integrin antibody (MAB1976 Millipore, Molsheim, France) diluted 1/200 in 1% BSA-supplemented PBS for 1 h at room temperature. After washing with PBS, cells were incubated respectively with a 1/1000 diluted Alexa fluor 488- or 568-conjugated secondary antibody (A11057 and A11004 respectively, Invitrogen, Carlsbad, USA) in PBS supplemented with 1% BSA for 30 min in a dark chamber at room temperature. Coverslips were then mounted with aqueous mounting medium (Thermo Scientific, Villebon sur Yvette, France) and examined using a confocal laser scanning microscope (Zeiss LSH710, Carl Zeiss MicroImaging, GmbH, Germany).

Standard single-color IHC was performed as described previously [17 (link)]. Briefly, 5 μm sections of formalin-fixed, paraffin-embedded tissue were deparaffinized and rehydrated by standard methods. Heat-induced epitope retrieval was performed in a 97°C water bath for 20 min in citrate buffer, pH 6.0 (for anti-CD1a, anti-CD3, anti-CD4, anti-CD16, anti-gp340 and their IgG negative controls), and the sections were re-equilibrated in PBS. Endogenous peroxidase and non-specific antibody binding were blocked (Ultra-V and Hydrogen Peroxide, LP Value kit), and then sections were incubated with respective primary antibodies at room temperature (30 min) or at 4°C (overnight), followed by detection with Enhancer, horseradish peroxidase (HRP)-Polymer, and diaminobenzidine tetrahydrochloride (DAB) substrate, all according to the manufacturer’s instructions (Fisher Scientific). All incubations, except non-specific protein binding (Ultra-V, LP Value Detection kit) block, were separated by washing with PBS. Coverslips were applied with aqueous mounting medium (Thermo Fisher Scientific) and photomicrographs were obtained (Olympus Corp., Tokyo, Japan). Each IHC run included sections stained with non-specific antibodies matched for species, isotype and concentration with the specific primary antibodies (negative controls).