Tcs sp2 laser scanning confocal microscopy

An inverted Leica TCS-SP2^® laser scanning confocal microscopy (Wetzlar, Germany) system was used to image cells adhering on the substrates. All measurements were performed using ArUv laser (Leica, Wetzlar, Germany). The pinhole was set to ≈ 80 μm (1.5 Airy units) and the laser power to 80% of the maximum, these values of the parameters were maintained constant throughout each acquisition. Confocal images of blue (DAPI) fluorescence were acquired using a 405 nm excitation line and a 10× dry objective, so that several cells could be simultaneously imaged in the region of interest, that was of 1174 × 882 μm², resulting in a pixel size of ≈ 1.72 μm. For each substrate, a large number of images was taken for statistical analysis. Each image was averaged over four lines and 10 frames to reduce noise. Images were acquired with a resolution of 1024 × 768 pixels, and were exported to a computer for processing and analysis.

Immunostaining for fluorescence microscopy was used to study the alteration of cytoskeleton and nuclei upon ZnO NPs exposure. At the end of treatments, samples were washed with PBS and fixed in 3.7% formaldehyde for 15 min. After removing formaldehyde, samples were washed twice in PBS, treated with 0.5% Triton X-100 for 5 min, and blocked in blocking solution (10% FBS, 1% bovine serum albumin in PBS) for 30 min. Microtubule staining was performed by using mouse monoclonal anti-α/β tubulin antibody mixture (Sigma Biosciences, Italy) diluted 1:50 in PBS with 1% BSA, followed by a mixture of secondary Alexa 488 goat anti-mouse IgG (1:50; Molecular Probes, USA) and phalloidin TRITC (1:100) to detect f-actin. Nucleus staining was carried out with PI (40 µg/ml) in PBS for 15 min. Cells were rinsed three times with PBS afterwards. Finally, samples were mounted in PBS containing 50% glycerol. Observations were performed by a Leica TCS SP2 laser scanning confocal microscopy (Leica, Microsystems, Mannheim, Germany) equipped with an Ar/Kr laser.