Tecnai 10 tem

For neutral BODIPY staining, 2 × 10⁵ RAW cells were seeded in each compartment of compartmented 35-mm sterile culture dishes with glass bottom (Cellview – Greiner). Cells were incubated for 4 h with AS03. Coverslips were washed and cells were stained with BODIPY 493/503 (Thermofisher) (1/500) and visualized with a Zeiss Axio Observer wide-field microscope.
For TEM, samples were washed with PBS and fixed with ice-cold glutaraldehyde 2% (EM grade, Sigma). Then, they were post-fixed in OsO₄ (2%) in 0.1 M cacodylate buffer (pH 7.2), serially dehydrated in increasing ethanol concentrations, embedded in Agar 100 resin (Agar Scientific Ltd, UK) and left to polymerize at 60°C for 2 days. Ultrathin sections (50–70 nm thick) were produced with a Leica EM UC6 ultra-microtome, collected on formvar-carbon-coated copper grids and stained with uranyl acetate and lead citrate by standard procedures. Observations were made on a Tecnai 10 TEM (FEI) and images were captured with a Veleta CCD camera and processed with SIS iTEM (Olympus).

Mel-Ab cells were treated with FSK for 72 h and prepared for electron microscopic analysis. Briefly, cultured cells were detached and fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde, followed by osmium tetroxide for 2 h. Two-month-old CTRL (C57BL6/J) and CRTC3-null mice tail tissues were also fixed in the same manner. Then, the fixed samples were dehydrated with ethanol and embedded in epon-araldite resin. Ultrathin sections were stained with 2% uranyl acetate and lead citrate and then examined by Tecnai 10 TEM (Fei, The Netherlands).