Fitc conjugated anti mouse igg secondary antibody

Cells were seeded at 1 × 10⁴ cells/well in a fourwell chamber slide (Nalge Nunc International, Rochester, NY). After 48 h incubation with graded concentrations of U0126 or LY294002, cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100/PBS for 15 min, and stored overnight at 4°C with 10% horse serum in PBS. The cells were washed and then incubated for 2 h with an anti- FASN mouse monoclonal antibody diluted 1:200 in 0.05% Triton X-100/PBS. After extensive washes, the cells were incubated for 45 min with FITC-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:200 in 0.05% Triton X-100/PBS. The cells were washed five times with PBS and mounted with VECTASHIELD + DAPI (Vector Laboratories, Burlingame, CA). As controls, cells were stained with primary or secondary antibody alone. No significant fluorescence was found in control experiments (data not shown). Indirect immunofluorescence was recorded on a Zeiss microscope. Images were noisefiltered, corrected for background, and prepared using Adobe Photoshop.

Cells were propagated in E₂-deprived (phenol red-free) IMEM with 5% CCS for 5 days before the onset of experiments. For experiments, MCF-7 were seeded at 1 × 10⁴ cells/well in a four-well chamber slide (Nalge Nunc International, Rochester, NY, USA). After 48 h incubation with E₂ in the absence or presence of tamoxifen, cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100/PBS for 15 min, and stored overnight at 4 °C with 10% horse serum in PBS. The cells were washed and then incubated for 2 h with an anti-FASN mouse monoclonal antibody diluted 1:200 in 0.05% Triton X-100/PBS. After extensive washes, the cells were incubated for 45 min with FITC-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA) diluted 1:200 in 0.05% Triton X-100/PBS. The cells were washed five times with PBS and mounted with VECTASHIELD + DAPI (Vector Laboratories, Burlingame, CA, USA). As controls, cells were stained with primary or secondary antibody alone. Indirect immunofluorescence was recorded on a Zeiss microscope. Images were noise-filtered, corrected for background, and prepared using Adobe Photoshop.