C2s confocal microscope

For confocal fluorescence microscopy imaging, T98G cells were seeded on μ-Plate 24 Well (Ibidi, seeding density of 2·10⁴ cells/cm²). At 12 h from seeding, cells were incubated for 24 and 72 h with 500 μg/mL Vybrant™ DiO-labeled PNPs and then fixed with 4% PFA in PBS for 20 min at 4°C. Cultures were then stained with TRITC-phalloidin (1:100 in PBS, Sigma-Aldrich) and Hoechst 33342 (1:1000 in PBS, Invitrogen) for 2 h. Samples were finally rinsed with PBS and imaged with a C2s confocal microscope (Nikon). 3D image reconstruction was finally performed by using NIS Elements Software (Nikon).
Concerning confocal Raman imaging, T98G cells were seeded on Raman grade calcium fluoride substrates (Crystran, seeding density 2·10⁴ cells/cm²). At 12 h from seeding, cells were incubated for 24 h with 500 μg/mL PNPs and then fixed with 4% PFA in PBS for 20 min at 4°C. Cultures were dehydrated and then imaged with a confocal Raman microscope (LabRAM HR Evolution, Horiba). Lab-Spec 6 software has been used to obtain the signal maps; signals of the PNPs (β-phase: 820 cm⁻¹ > Raman shift > 880 cm⁻¹), cell proteins (amide I region [46 (link)]: 1600 cm⁻¹ > Raman shift > 1700 cm⁻¹), and nuclei (DNA [47 (link),48 (link)]: 760 cm⁻¹ > Raman shift > 790 cm⁻¹) were used, with a pixel intensity proportional to the integrated peak area.

The spheroids were fixed with 4% paraformaldehyde for 20 min, and after 30 min of incubation with the blocking solution (Triton 100 × 1:1000, 10% goat serum in PBS), they were incubated for 3 h with primary anti-Ki-67 antibody (1:150 dilution; Millipore) at 37 °C. The secondary FITC-conjugated goat anti-rabbit antibody (1:250 dilution; Millipore) was administered for 2 h and, after the washing steps, staining with TRITC-phalloidin (1:200 dilution; Sigma-Aldrich) and Hoechst 33342 (1:1000 dilution; Invitrogen) in PBS was carried out for 1 h at 37 °C. Images were acquired with a C2s confocal microscope (Nikon).