Cy3 conjugated anti mouse

To indirectly determine the expression levels of FTH1 by immunostaining, Flag-expressing SK-N-SH-FTH1 cells were treated under the same conditions as those of the western blot analysis. After treatment, the cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and thoroughly washed. The samples were permeabilized with 1% Triton X-100 in PBS, blocked with 5% BSA at 37°C for 30 min, and incubated overnight at 4°C with a primary antibody against Flag (mouse anti-Flag 1:200; Abgent, San Diego, CA, USA). The cells were then washed with PBS and incubated in the dark at room temperature with the secondary antibody (Cy3-conjugated anti-mouse, 1:1,000; Beyotime, Nanjing, Jiangsu, China). Subsequently, 4, 6-diamidino-2-phenylindole (DAPI; Beyotime, Nanjing, Jiangsu, China) was used to counterstain cell nuclei. After mounting coverslips, the samples were imaged using a fluorescence microscope (Nikon, Tokyo, Japan).

The Flag tag was used to indirectly determine the expression levels of FTH1 via immunocytochemistry using a Flag-specific antibody. C3H10T1/2-FTH1 cells were cultured for 72 h in the same concentrations of Dox as those used in the western blot experiments. Then, the cells were fixed in 4 % paraformaldehyde (PFA) for 15 min at room temperature. The fixative solution was removed, and the cells were washed with PBS three times for five min each. The cells were permeabilized with 1 % Triton X-100 in PBS for ten min, blocked with 5 % BSA in PBS at 37 °C for 30 min to 1 h, and then incubated in a specific primary antibody (mouse anti-Flag 1:200; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C. After three washes with PBS, the cells were incubated in a secondary antibody (Cy3-conjugated anti-mouse, 1:1,000; Beyotime, Nanjing, Jiangsu, China) for 30-45 min at 37 °C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, Nanjing, Jiangsu, China) for five min. Images were acquired using a biological fluorescence microscope (Nikon, Tokyo, Japan).