Laser scanning microscopy showing LD labeling of perilipin proteins in briefly AIM-stimulated human preadipocytes which were additionally treated shortly with oleic acid (OA).
(a, a, a) AIM treatment for 1–3 days reveals many newly generated small and medium-sized LDs positively staining with perilipin antibodies (red). Additional OA-treatment for 2 hours leads to the appearance of LDs positive for adipophilin (green) in higher numbers and bigger sizes when compared to non-stimulated or AIM-stimulated cells solely (cp. Fig. 1). (a) Enlarged part taken from double staining seen in (a). (a) Enlarged part from (a) showing single color and mosaic double-stained droplets, obviously in the midst of a fusion process with involvement of different types of LD-binding PLIN proteins and with yellow, partly mixed colored LDs for co-localization. (b) A different perilipin/adipophilin antibody combination reveals a similar heterogeneous staining of LDs as seen in a-a. (c) Perilipin (red) vs. S3-12 (green) double staining. Several smaller S3-12-positive droplets are seen attaching and combining with larger perilipin-positive droplets. Many newly generated, very small and middle-sized rings of S3-12-positive droplet staining with punctate patterns can be seen by single green color staining only (examples marked by arrowheads). (d) Perilipin (red) vs. TIP47 (green) double staining. Some of the many newly appearing TIP47-positive small droplets adhere occasionally to the much bigger perilipin positive droplets. (e) Adipophilin (red) vs. TIP47 (green) double staining. Most of the droplets are visible with sizes estimated smaller than 1 µm in diameter. Some heterogeneously colored droplets are marked by arrowheads. Note, OA-treatment leads to the re-appearance of a tremendous number of small droplets positive for adipophilin, TIP47 and S3-12. Note in addition, the complexity of the PLIN staining of LDs. Different LD-binding proteins are expressed and different types of LDs appear in single cells by specific “endogenous” and “exogenous” stimulations. DAPI (blue). Bar in a: 5 µm; all other Bars: 20 µm.