Spot rt3 camera

Dorsal skin or tumor samples were fixed in 4% paraformaldehyde in PBS, dehydrated in ethanol, embedded in paraffin, and sectioned at 5 μm. For immunofluorescence detection, sections were deparaffinized, rehydrated, and subjected to antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0). After blocking, sections were stained with primary antibody at 4 °C overnight in a humid chamber, followed by incubation with the appropriate fluorochrome-conjugated secondary antibody for 2 h at room temperature. Nuclei were stained with Hoechst 33342 dye (Sigma, St. Louis, MO, USA). Immunohistochemistry was performed on paraffin-embedded sections subjected to antigen retrieval as described above. After blocking, sections were incubated with primary antibodies at 4 °C overnight in a humid chamber, followed by incubation with biotinylated IgG at RT for 30 min. Detection of the signal was carried out using Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA) according the manufacturer’s instructions. The sections were counterstained with hematoxylin. Microscopy was performed using a fluorescence microscope (Nikon Eclipse E600), Spot RT3 camera and Spot imaging software.

NRCM cells were seeded at a density of 10⁵ cells/well in 6 well plates on glass coverslips with 1% gelatin coating and cultured at least for 2 days before the experiment. On the day of the experiment, cells were washed once in PBS and added fresh medium and, then, treated with H₂O₂ with or without BGP-15. After the appropriate treatment, coverslips were rinsed in PBS and were added 75 nM MitoTracker Red CMXRos dissolved in serum-free DMEM and incubated for 30 min at 37°C. After the incubation, coverslips were rinsed in PBS, and the mitochondrial network was visualized by a Nikon Eclipse Ti-U fluorescent microscope equipped with a Spot RT3 camera using a 60x objective and epifluorescent illumination.