Fv1200mpe multiphoton laser scanning microscope

The S30 and unexposed BEAS-2B cells were fixed in PBS containing 4.0% paraformaldehyde without methanol. The cells were washed and permeabilized with 0.2% Triton X-100 and blocked with 5% goat serum for 1 hour at room temperature. Diluted antibodies for human NME1 (11086-2-AP, Proteintech, Chicago, IL, USA), SLIT2 (20217-1-AP, Proteintech, Chicago, IL, USA) or GYPC (ab108619, Abcam, Cambridge, MA, USA) were added drop by drop and the slides were kept in a wet box at 4 °C overnight. Following incubated with FITC-conjugated goat anti-rabbit IgG for 1 hour at room temperature, the slides were washed, and the nuclei were counter-stained with 4,6-diamidino-2-phenylindole (DAPI). Photographs were taken and visualized using an FV1200MPE multiphoton laser scanning microscope (FV1200, OLYMPUS, Japan). The acquisition parameters were held constant for all the experiments.

An Olympus FV1200 MPE multiphoton laser scanning microscope monitored the presence of nanoparticles inside the rMSCs and C6 cells. Briefly, the cells were fixed with 4% paraformaldehyde in PBS and stained with DAPI. Subsequently, brightfield images were taken at 980-nm excitation and 540-nm emission using an IR pulsed laser with negative chirp for multiphoton excitation. The DAPI-stained cells were visible in blue channel with laser diode at 405 nm.