Lab tek 2 cc2 8 well chamber slide system

After transfection or treatments, cells were seeded in a Nunc Lab-Tek II CC2 8-well Chamber Slide System at a density of 10,000/well and cultured for 2 days. Cells were fixed with 4% paraformaldehyde or with ice-cold methanol for 15 min. Samples were blocked in PBS/5% BSA/0.3% Triton X-100 for 1 h and incubated overnight at 4 °C with TGFBR1 (ab235178, 1/100, Abcam) or BMPR2 (ab130206, 1/100, Abcam). After washing in PBS, cells were incubated with Alexa Fluor™ 568 goat anti-mouse or anti-rabbit secondary antibodies (1/500, Life Technologies, Thermo Fisher Scientific). Slides were mounted using Fluoromount-G medium (#00-4958-02; Thermo Fischer Scientific). Images were acquired using a LEICA DMI 4000B confocal microscope (Leica Microsystems SA, Nanterre, France) with a 63× magnification oil-immersion objective. The intensity of fluorescence was measured using ImageJ software.

After treatment, cells were seeded in the Nunc Lab-Tek II CC² 8-well Chamber Slide System at a density of 2000 per well and cultured for 2 days. Cells were then fixed in ice-cold methanol for 10 min and washed in PBS. After a 60 min blocking step in PBS/3% BSA/0.3% Triton X-100, cells were incubated overnight at 4 °C with EGFR antibody (#4267; Cell Signaling Technology, Ozyme, Saint-Cyr-L’Ecole, France; dilution 1/50). After washing in PBS, cells were incubated with appropriate secondary antibodies (Life Technologies; dilution 1/500) and DAPI (#D9542; Sigma-Aldrich, St Quentin Fallavier Cedex, France; 1 µg/mL). After washing in PBS, the slides were mounted using Fluoromount-G medium (#00-4958-02; Thermo Fisher Scientific, Illkirch, France). Images were acquired using a LEICA TCS SPE II confocal microscope (Leica Microsystems SA, Nanterre Cedex, France), with a 60 × magnification oil-immersion objective, and analyzed with ImageJ software (https://imagej.nih.gov, access on 3 May 2021).