Anti rabbit igg fluorescein isothiocyanate fitc conjugated secondary antibody

U87MG and GBM8401 cell lines pre-treated with or without 2.5 μM or 5 μM of garcinol were seeded in 6-well chamber slides (Nunc, Thermo Fisher Scientific, Taipei, Taiwan), incubated at 4 °C overnight, fixed with 2% paraformaldehyde for 10 min at room temperature, and permeabilized in 0.01 M phosphate-buffered saline (PBS) with 0.1% Triton X-100 and 0.2% bovine serum albumin (BSA). The slides were air-dried and rehydrated with PBS before incubation with primary antibodies against OCT4 (#2840; Cell Signaling Technology) and/or SOX2 (#3579; Cell Signaling Technology) at 1:500 dilution in PBS for 2 h at room temperature. The slides were washed with PBS twice for 10 min each, then incubated with anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated secondary antibody (diluted 1:500; Jackson Immunoresearch Lab. Inc., West Grove, PA, USA) in PBS for 1 h. The slides were mounted with Vectashield mounting medium and counter stained with 4′, 6′-diamidino-2-phenylindole (DAPI) for nucleus visualization.

For the immunofluorescence analysis, the OSCC cells were plated in 6-well chamber slides (Nunc™, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 24 h, fixed in 2% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 in 0.01 M PBS (pH 7.4) containing 0.2% BSA, air-dried, and rehydrated in PBS. The cells were then incubated with antibody against CD47, Oct4, Sox2, c-Myc, vimentin, or E-cadherin diluted 1:200 in PBS containing 3% normal goat serum at room temperature for 2 h, followed by incubation with anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Jackson ImmunoResearch Lab. Inc., West Grove, PA, USA). The cells were allowed to rest at room temperature for 1 h, washed in PBS, and mounted using Vectashield mounting medium while counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, D3571, Molecular Probes, Life Technologies Co., Carlsbad, CA, USA). Images were captured using a Zeiss Axiophot (Carl Zeiss Co. Ltd., Hsinchu City, Taiwan) fluorescence microscope, and the microphotographs were analyzed using ImageJ software v. 1.46 (https://imagej.nih.gov/ij/).