Tcs sp5 confocal

Cells were seeded in 8 wells-chamber slides and stimulated with PA, oleate, DHA or SU6656. After the indicated incubation time, cells were washed with PBS and fixed for 30 min in PBS containing 4% paraformaldehyde and 2% sucrose, followed by three washes in PBS. Slides were incubated in PBS with Triton-X-100 (PBST) for 5 min at 4°C to permeabilize the cells. Cells were washed and blocked in PBST with BSA 5% for 1 h at room temperature and incubated with either Fyn or Nrf2 antibody diluted 1:200 in PBST-BSA 2%, followed by Alexa Fluor 488 anti-rabbit IgG or IgG or Alexa Fluor 594 anti-goat IgG for 1 h at room temperature. Samples were mounted with Prolong Gold anti-fade reagent with DAPI (Life Technologies). Fluorescence images were acquired using a confocal fluorescence microscope (TCS SP5 confocal; Leica microsystems, Buffalo Grove, IL, USA).

3T3L1 adipocytes were transfected with either pcDNA3-Flag-LKB1-WT or mutants (150 µg), washed with PBS and fixed for 10 min in PBS containing 4% PFA and 0.2% Triton X-100. Immunofluorescence was performed using a Flag monoclonal antibody followed by Alexa Fluor 488. Samples were mounted on glass slides with Prolong Gold anti-fade reagent with DAPI (Life Technologies, Grand Island, NY, USA). Cells were imaged using a confocal fluorescence microscope (TCS SP5 confocal; Leica microsystems, Buffalo Grove, IL, USA).