Fitc conjugated anti rabbit secondary antibodies

Cells were seeded in BD Falcon™ 96-well black, clear-bottom tissue culture plates at 10,000 cells per well. These plates are optimized for imaging applications. Analyses were performed in duplicate in three independent experiments. After incubation, cells were rinsed with PBS and fixed with a 3.7% formaldehyde solution at room temperature for 10 min. Cells were then washed three times with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. Next, the cells were washed twice with PBS, and nonspecific binding was blocked by incubation in 3% FBS at room temperature for 30 min. The cells were rinsed and incubated with either anti-Nrf2 rabbit polyclonal antibodies (Sigma-Aldrich, St. Louis, MO, USA; 1 : 1000) or anti-NFκB (p52) mouse polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Cells were then washed three times with PBS and incubated with FITC-conjugated anti-rabbit secondary antibodies (BD Pharmingen, San Diego, CA) for 60 min in the dark. After washing, nuclei were stained with Hoechst 33342 (2 μg/mL) and analyzed using a BD Pathway 855 confocal microscope with a 40x (0.75 NA) objective. The cytoplasmic and nuclear fluorescence intensities of stained cells were analyzed, and images of FITC-labeled cells were acquired using a 488/10 excitation laser and a 515LP emission laser.

The cells were seeded in BD Falcon™ 96-well black, clear-bottom tissue culture plates (BD Biosciences, San Jose, CA, USA) at 10,000 cells per well. After incubation, L6 cells were rinsed with PBS and fixed with a 3.7% formaldehyde solution (Sigma Aldrich) at room temperature for 10 min. Then, they were washed three times with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. Afterwards, they were washed twice with PBS, and non-specific binding was blocked by incubation in 3% FBS at room temperature for 30 min. The cells were rinsed and incubated with anti-CD36/SR-B2, -FABPpm, -FATP1, -FATP4, -GLUT4, and -PGC-1α primary antibodies for night at room temperature. Next, they were washed three times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies (1:500; BD Biosciences) for 60 min in the dark. After washing, nuclei were stained with Hoechst 33342 solution (2 μg/mL, blue) and analyzed using a BD Pathway 855 confocal microscope with a 40 × (0.75 NA) objective. The fluorescence intensities of the stained cells were evaluated, and images of FITC-labeled cells were acquired using a 488/10 excitation laser and a 515LP emission laser.

Cells were seeded in BD Falcon™ 96-well black, clear-bottom tissue culture plates at 10,000 cells per well. These plates are optimized for imaging applications. Analysis were performed in three independent experiments. After incubation, cells were rinsed with PBS and fixed with a 3.7% formaldehyde solution at room temperature for 10 min. Cells were then washed three times with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. Next, the cells were washed twice with PBS, and non-specific binding was blocked by incubation in 3% FBS at room temperature for 30 min. The cells were rinsed and incubated with either anti-Nrf2 rabbit polyclonal antibodies (Sigma–Aldrich, St. Louis, MO, USA; 1:1000) or anti-NFκB (p52) mouse polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Cells were then washed three times with PBS and incubated with FITC-conjugated anti-rabbit secondary antibodies (BD Pharmingen, San Diego, CA) for 60 min in the dark. After washing, nuclei were stained with Hoechst 33,342 (2 µg/ml) and analyzed using a BD Pathway 855 confocal microscope with a 40 × (0.75 NA) objective. The cytoplasmic and nuclear fluorescence intensities of stained cells were analyzed, and images of FITC-labeled cells were acquired using a 488/10 excitation laser and a 515LP emission laser.