Dapi containing medium

4 µm sections were serially dewaxed 3×10 min in Histochoice clearing agent (Sigma), 2×5 min in 100% ethanol, and 3 min in 90%, 70% and 50% ethanol. Sections were then immersed in water for at least 5 min and boiled in citrate buffer (pH 6.0) for 2 min. Next, sections were rinsed in PBS and blocked overnight with 30% FCS in PBS 0.1% Triton X-100. Sections were then incubated with the corresponding primary antibodies in PBS 10% FCS: Rabbit anti mouse CD31 (1∶100, Abcam), rabbit anti mouse VE-cadherin (1∶800, Abcam) or monoclonal Cy3-conjugated antimouse α-SMA (1∶1000, Sigma). CD31 was detected using the DAB method (Biogenex), whereas VE-cadherin was incubated in cy3-conjugated goat anti rabbit (1∶500, Life Technologies). For immunocytochemistry, MEECs and MEFs were cultured for two days in gelatin coated coverslips, fixed for 15 min in 4% PFA, permeabilized for 10 min with PBS 0.25% Triton X-100, blocked in PBS 10% FCS and 0.05% Tween 20 (PBST) for one hour at room temperature, incubated with PBST containing rabbit antimouse CD31 (1∶50, Abcam) overnight at 4°C, washed 3×10 min in PBST, and incubated with cy3-conjugated goat anti-rabbit (1∶500, Life Technologies) for two hours at room temperature. Cells were photographed upon mounting with Dapi containing medium (Life Technologies).

3T3-L1 cells were grown on 0.2% gelatin-coated coverslips and induced to differentiate into adipocytes with standard DMI. At the indicated time point cells were fixed with 95% ethanol, followed by permeabilization with 0.1% Triton X-100 and 0.5% NP-40 on ice. After blocking with 5% nonfat dried milk in TBST, the coverslips were incubated with primary antibody, namely, mouse anti-METTL3 (Abnova), rabbit anti-METTL14 (Sigma), goat anti-WTAP (Santa Cruz), mouse-anti-SC-35 (Abcam), or mouse anti-Smith antigen (Thermo Fisher Scientific), overnight at 4°C. The coverslips then were incubated with fluorescent dye-conjugated secondary antibody, namely, anti-mouse IgG–Alexa Fluor 488 (Cell Signaling Technology), anti-rabbit IgG–Alexa Fluor 594, or anti-goat IgG–Alexa Fluor 647 (Abcam), for 0.5 h at 37°C and mounted with DAPI-containing medium (Life Technologies). Fluorescent images were acquired using an FV10i confocal microscope (Olympus).