Alexa fluor 594 labeled goat anti mouse igg

After attaching to the bottom of 4-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA), FRTL-5 cells were maintained in the absence of TSH for 5 days. Cells were then treated with 1 mU/mL TSH for 6, 12, 24, 48 and 120 h, and 1 μM forskolin or 500 μM dbcAMP for 120 h. After incubation, cells were fixed using 10% neutral buffered formalin for 10 min at room temperature, and then permeabilized with PBS containing 0.1% Triton X-100. After blocking with PBS containing 3% bovine serum albumin, the slides were incubated with mouse anti-Slc26a7 (1:500; Novus Biologicals) overnight, washed with PBST and then incubated with Alexa Fluor 594-labeled goat anti-mouse IgG (1:1,000; Cell Signaling Technology), followed by nuclear staining with Hoechst 33342 (Cell Signaling Technology). Fluorescence was observed under a confocal laser scanning microscope (FV10i-DOC, Olympus, Tokyo, Japan).

After attaching to the bottom of 4-well chamber slides (Thermo Fisher Scientific), FRTL-5 cells were maintained in the absence of TSH for 5 days. Cells were then treated with 1 mU/mL TSH and/or 5 mg/mL Tg. Immunofluorescence staining was performed as described previously [12] . Briefly, cells were fixed using 10% neutral buffered formalin for 10 min at room temperature and then permeabilized with PBS containing 0.1% Triton X-100. After blocking in PBS containing 3% BSA, the slides were incubated with mouse anti-SLC26A7 (1:500; Novus Biologicals) overnight at 4°C, washed with PBST, and then incubated with Alexa Fluor 594-labeled goat anti-mouse IgG (1:1,000; Cell Signaling Technology), followed by nuclear staining with Hoechst 33342 (1:1,000; Immunochemistry Technologies, Bloomington, MN, USA) for 10 min at room temperature. Fluorescence was observed under the FV10i-DOC confocal laser scanning microscope (Olympus, Tokyo, Japan).