Ti2 a1rhd25

Cells were fixed in paraformaldehyde 4% (Sigma Aldrich, Saint-Louis, MO, USA) and blocked in PBS 1x (Klinipath, Olen, Belgium) containing 0.3% TritonTM X-100 and 5% normal goat serum (Cell Signaling Technology). The cells were incubated overnight at 4 °C with antibody anti-ATP1A1 (M7-BP-E9, mouse, dilution 1:400) (ThermoFisher Scientific) and anti-Cav1 (3238, rabbit, dilution 1:200) (Cell Signaling Technology) diluted in PBS 1x, 0.3% TritonTM X-100 and 1% BSA (Sigma). The secondary antibodies were, respectively, Alexa FluorTM 555 Goat AntiMouse IgG and Alexa FluorTM 488 Goat Anti-Rabbit IgG (Invitrogen). Nucleus were stained using VectaShield-DAPI (Vector laboratories). Images were acquired using a confocal microscope (Nikon Ti2 A1RHD25, Tokyo, Japan).

Monocytes were polarized in macrophages on the coverslip of 24-well plates. When macrophages were differentiated in M1 and M2, cells were fixed for 15 min (10 min at 4 °C followed by 5 min at room temperature) with 4% paraformaldehyde (Sigma-Aldrich) in PBS, and then rinsed with PBS. For CD68 labeling, rinsing was followed by a permeabilization step in cold methanol for 10 min at −20 °C. Next, cells were incubated with a blocking solution, followed by antibody incubation (Table 3). After 3 washes, cells were incubated with the secondary antibody (1/500 dilution in blocking solution) for 1 h. The coverslips were then rinsed with PBS and distilled water before being mounted on slides with VectaShield-DAPI (VectaShield, Vector laboratories, Newark, CA, USA). Once dried, the slides could be observed under a confocal microscope (Nikon Ti2 A1RHD25, Tokyo, Japan).

On the 7th day of coculture, spheroids were washed in PBS and fixed with paraformaldehyde 4% (Sigma-Aldrich, St. Louis, MI, USA) overnight at 4 °C and then rinsed in PBS. A permeabilization step is required overnight with 0.5% triton X-100 at 4 °C followed by washing steps with PBS. Next, spheroids were incubated with a blocking solution for 2 h followed by antibody incubation for 24 h (Table 7). The next day, spheroids were incubated with the secondary antibody (1/500 dilution) for 1 h followed by rinsing with PBS and distilled water. Then, Hoechst dye (BisBenzimide Sigma Aldrich) was used for 15 min in order to stain nuclei in spheroids. Finally, they were transferred onto a coverslip and mounted on the slides with Vectashield (Vector laboratories, Newark, CA, USA). Once they were dried, slides were observed under a confocal microscope (Nikon Ti2 A1RHD25, Tokyo, Japan). Quantification of fluorescence intensity was examined with QuPath software Version 0.4.3.