Eclipse te2000

Reactive oxygen species (ROS) in embryos were evaluated with dihydroethidium (DHE) fluorescent staining as in a previously described method [22 (link)]. In brief, zebrafish embryos (n = 20) at 5 h post-treatment were stained with DHE (30 μM). After 30 min incubation in the dark, embryos were rinsed twice with 1 × PBS and visualized under a fluorescent microscope (Nikon Eclipse TE2000, Tokyo, Japan) at the excitation and emission wavelengths of 588 nm and 605 nm, respectively. The apoptosis in the embryos was examined by acridine orange (AO) fluorescent staining [22 (link)]. At 5 h post-treatment, zebrafish embryos (n = 20) from different groups were suspended in 500 μL of AO (5 μg/mL). After 1 h staining, embryos were rinsed two times with 1 × PBS and visualized under a fluorescent microscope (Nikon Eclipse TE2000, Tokyo, Japan) at the excitation and emission wavelength of 502 nm and 525 nm, respectively. Image J software was employed for quantifying the fluorescently stained area.

The method described earlier [23 (link)] was employed to assess the production of reactive oxygen species (ROS) in the tissue section (n = 5 for each group) using dihydroethidium (DHE) fluorescent staining. Briefly, a 250 μL of DHE solution (final, 30 μM) was added to the tissue section. Following 30 min incubation in the dark at room temperature (RT), the stained area was rinsed with tap water and observed using a fluorescent microscope (Nikon Eclipse TE2000, Tokyo, Japan) with excitation at 588 nm and emission at 618 nm wavelength. The extent of apoptosis was examined using acridine orange (AO) fluorescent staining, following a method described previously [24 (link)] with slight modification. In summary, a tissue section (n = 5 for each group) was treated with 250 μL of AO (5 μg/mL). After 30 min incubation in the dark at RT, the stained section was washed two times with tap water and subsequently visualized under a fluorescent microscope (Nikon Eclipse TE2000, Tokyo, Japan) at an excitation wavelength of 505 nm and an emission wavelength of 535 nm.

To estimate the genotoxic effect of the investigated nanoparticles, we used single-cell gel electrophoresis (SCGE) in the form of alkaline comet assay according to the procedure of [56 (link)]. In our experiments we applied the Comet Assay Kit (Trevigen, Minneapolis, MN, USA). The cells were plated onto a 24-well transparent plate. After 24 h the cells were treated with nanoparticles for a further 24 h. After this time, the cells were trypsinized and suspended in low melting point agarose in PBS, pH 7.4. 50 mL of cell suspension was spread on microscope slides supplied in Comet Assay Kit. After gelling, the slides were treated with lysis buffer containing 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10% DMSO and 10 mM Tris, pH 10 at 4 °C for 1 h. Slides were then placed in the electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH > 13) for 40 min. Electrophoresis was carried out at 0.73 V/cm, 300 mA for 30 min. The slides were then neutralized, stained with 2 mg/mL DAPI and analyzed under fluorescent microscope (Nikon Eclipse TE2000, Nikon, Tokyo, Japan). All the steps of this procedure were performed in the dark. The level of DNA damage was determined on the basis of the comet tail moment using CaspLab software (1.2.3beta1 version, Comet Assay Software Project, Wrocław, Poland) [57 (link),58 (link)].