Spinning disk microscope

Rptor^fl/fl MPMECs were transduced with Ad-CMV-Null or Ad-CMV-iCre for 24 hrs on MatTek dishes with a No. 1.5 coverslip coated with 0.1% gelatin. Cells were stimulated with VEGF-B (300 ng/mL) for 30 hrs in EBM-2 supplemented with 1% FFA-BSA. Prior to imaging, live cells were stained with Phalloidin-iFluor594 (Abcam #ab176757) and Hoescht 33342 (Invitrogen #R37605), according to manufacturer’s instructions. Cells were incubated with BODIPY FL C16 (20 μM) for 20 min at 4°C, then washed 3x in EBM-2 with FFA-BSA. Cells were then incubated at 37°C for 5 min and immediately imaged using a Nikon Spinning Disk Microscope. Images from three regions were collected from three independent experiments. The z-plane representing the apical (top) and basolateral (bottom) edges of the cell were determined by phalloidin staining. The basolateral surface represents the bottom 10% of z-plane images for each cell. BODIPY-C16 intensity was quantitated in ImageJ, and the intensity present at the basolateral surface was calculated as a percent of whole cell intensity.

~13 larvae per condition or genotype were vortexed and bled into 300 μl of Schneider’s insect media and then the cells were transferred to one well (per condition) of the Lab-Tek II chambered coverglass (VWR, cat# 62407-056). Hemocytes were allowed to settle down for 30 min, then were fixed in 4% paraformaldehyde (PFA) for 20 min. Next, the cells were washed three times with 1X PBS and permeabilized with 0.1% PBST (PBS with 0.1% Triton-X) for 10 min. Subsequently, the cells were blocked with 5% BSA in PBST (blocking buffer) for 20 min and subsequently incubated with primary antibodies L1abc (1:100 dilution) overnight at 4°C. The next day, cells were washed three times in PBST and incubated with corresponding secondary antibodies (1:500 dilution) for 1 hr at room temperature. Finally, the cells were washed (3X) and mounting media with DAPI was directly added onto the cells in the wells and imaged using Nikon Spinning disk microscope.

Cells were seeded to reach 40%–60% confluence in 96-well or 24-well glass bottom plates (Cellvis, Mountain View, USA) coated with poly-L-lysine (Sigma, P4707). After washed with PBS, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed with PBS for three times. Next, fixed cells were incubated in blocking solution (containing 5% (v/v) Normal Goat Serum (Bioss Antibodies, C01-03001), 0.3% (v/v) Triton X-100 in PBS) for 2 h at room temperature. After that, the cells were incubated with primary antibody of RXRα (Santa cruz, sc-515929), PPARγ (Cell Signaling, 2435 S) or mCherry (Thermo Fisher scientific, M11217) overnight at 4 °C. Next, cells were washed three times in PBS and then incubated with secondary antibodies conjugated to Alexa Fluor 555 (Cell Signaling, 4409 S), Alexa Fluor 488 (Cell Signaling, 4412 S) or Alexa Fluor Plus 488 (Thermo Fisher scientific, A48262) at 1:1000 dilution for 2 h at room temperature. During this period, 96-well plate was wrapped in foil to keep it in dark environment. Cells were then washed three times in PBS for 10 min. Nuclei staining was performed with DAPI (YEASEN, 40728ES10). Images were acquired at the Nikon Spinning Disk microscope with 100× oil immersion objectives. Fluorescence intensity was obtained using FIJI (National Institutes of Health, Bethesda, USA).