Fluorview fv500 confocal microscope

Actin cytoskeleton integrity was evaluated only in hemocytes according to Chazotte [43 ]. After in vitro exposures, hemocytes were fixed with methanol for 10 min, permeabilized with acetone for 20 sec and incubated with tetramethylrhodamine B isothiocyanate (TRITC)-conjugated phalloidin for 30 min in the dark. Cells were then washed several times with PBS, and observed under an Olympus Fluorview FV500 confocal microscope (Hamburg, Germany) at excitation 540-545/emission 570–573 nm.

To evaluate internalization and subcellular localization of selected PS-MSN samples through confocal microscopy, cells were incubated for 24 h with 1 μM of RB, RB-PEG-NP-d or BDP6-NP in 10% FBS-supplemented DMEM culture medium. Unexposed cells were used as control. After exposures, cells were washed three times with culture medium and fixed with 0.4% paraformaldehyde for 10 min at 4 °C. Cells were then washed three times with culture medium and observed under an Olympus Fluorview FV500 confocal microscope (Hamburg, Germany). Fluorescence images were obtained under 561 nm excitation and 565–616 nm emission range (Alexa 568) for RB samples and at 640 nm excitation and 645–700 nm emission range for BDP-6 sample. Both fluorescence and brightfield images were obtained at the same depth. Images were edited using Fiji software (ImageJ 1.49a, National Institutes of Health, Bethesda, MD, USA).