Poly l lysine coated culture coverslips

FRTL-5 cells were grown on poly-L-lysine-coated culture coverslips (Matsunami Glass, Osaka, Japan) in a 24-well plate and treated with 1 mg/mL bovine thyroglobulin (Tg) (Sigma Aldrich) and 5 mM MMI or PTU for 24 hours. To label lysosomes, LysoTracker Red DND-99 (Life Technologies, Carlsbad, CA, USA) was added to the culture medium at a final concentration of 50 nM and incubated with the cells at 37°C for 1 hour. Cells were fixed with 10% buffered formalin (Wako Pure Chemical) for 15 minutes, permeabilized with 0.3% Triton X-100 (Wako Pure Chemical) in Dulbecco ' s Phosphate-Buffered Saline (DPBS) for 5 minutes, and blocked with 0.5% bovine serum albumin (Sigma Aldrich) in DPBS for 1 hour. Immunofluorescence staining of Dehal1 was performed by incubating the coverslips with rabbit polyclonal anti-Iyd (1:500; Abcam) at 4°C overnight. Coverslips were incubated with Alexa Fluor 488-conjugated chicken anti-rabbit IgG antibody (1:1,000; Life Technologies) for 1 hour at room temperature. The nuclei were counterstained with Hoechst 33258 (1:1,000, Life Tech-nologies), and coverslips were placed on glass slides with fluorescence mounting medium (Dako, Tokyo, Japan). Immunofluorescence was visualized, and the images were captured using the FV10i-LIV confocal laser scanning microscope (Olympus, Tokyo, Japan).