Super-Resolution Imaging of PLIN1 in Adipocytes

High-resolution fluorescence microscopy by dSTORM imaging was performed on a TIRF-based Nikon N-STORM Super-Resolution microscope system according to published protocols^{20 (link),23 (link)}. All imaging was performed using the SR Apochromat TIRF 100 × 1.49 N.A. objective lens. We used the MEA-based imaging buffer as previously described, consisting of 10 mM 2-aminoethanethiol (MEA; Fluka) in 50 mM Tris, pH 8.0, 10 mM NaCl, 10% (w/v) glucose, and 0.5 mg/ml glucose oxidase (Sigma-Aldrich) and 40 μg/ml catalase (Sigma-Aldrich) as the oxygen scavenger system^{20 (link)}. PLIN1 in human primary adipocytes was detected by immunofluorescence staining using a primary PLIN1-specific primary antibody and the Alexa Fluor® 647 conjugated secondary antibody. This approach can visualize cellular structures with a resolution of approximately 20 nm, which is well below the Abbe diffraction limit^{22 (link)}. Another advantage with this approach is that the adjustable TIRF angle only penetrates to a depth of only approximately 100 nm into the sample medium, which enables selective visualization of surface regions (reviewed in ref.^{20 (link)}). The Nikon NIS-Elements software was used for data acquisition, image processing and analysis.