Ab119501

Immunostainings using isolectin B4 (Vector Laboratories, California, USA) were performed according to standard procedures (22). For immunostainings using primary antibody recognizing perilipin (#9349, Cell Signaling) or resistin (ab119501, abcam, Cambridge, UK) cells were washed 3 times with PBS and blocked with 10% donkey serum and 0.3% Triton in PBS for 1 h at room temperature. Cells were stained with perilipin antibody (1:100) or resistin antibody (1:50) overnight at 4°C. Cells were washed 3 times, and incubation with the secondary antibody Cy3-anti-rabbit (1:250, Jackson ImmunoResearch) was performed for 2 h at room temperature. Nuclei were stained with DAPI Hoechst 33342 (Sigma-Aldrich). Images were acquired with AxioVert200M microscope and Axiovison software 4.8, Axio Observer 7, and Zen 2.6 pro software (Carl Zeiss).

Equal amount (50 μg) of protein for each sample was loaded and separated on a 10% SDS-PAGE. After electrophoretic separation, the proteins were transferred onto PVDF membranes. According to each target protein, the membrane was incubated with mouse monoclonal antibody to adiponectin (mouse monoclonal antibody, ab22554, Abcam) or rabbit polyclonal antibody to resistin (ab119501, Abcam) or mouse monoclonal antibody to resistin (ab136877, Abcam), at 4°C for overnight. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, immunoreactive protein bands were visualized using the enhanced chemiluminescence detection substrate photoreactive X-ray films. Densitometry quantification of protein band intensity was performed using the TINA software (Raytest, Straubenhardt, Germany). The expressions of each target protein were present as fold of the loading control, β-actin.