Laser scanning single- and double-label immunofluorescence microscopy showing lipid droplet (LD) labeling of perilipin proteins in untreated, non-stimulated and in briefly AIM-stimulated human preadipocytes.
(a-c) Non-stimulated cells. (a) Adipophilin monoclonal antibody (mab) staining reveals many small LDs (green). (b) Positive TIP47 polyclonal antibody (pab; green) vs. negative perilipin (mab; red) staining. (c) Pab S3-12 shows many very small LDs – some can be seen like arranged and placed along rows of filaments. (d-g) Adipocyte differentiation of cells with AIM medium. 1-3 days of AIM treatment newly generates many small and medium-sized LDs, which stain with antibodies for perilipin (red). In contrast after AIM treatment, LDs positive for other perilipin (PLIN) family members are reduced in numbers and size (green). Cells still appear fibroblast-like elongated, not roundish. (d) Perilipin vs. adipophilin staining. (e) Perilipin and TIP47 double staining. (f) Perilipin localization at surface of LDs (g) Perilipin comparison with S3-12. Note, whereas antibodies specific for adipophilin, S3-12 and TIP47 show plenty of small LDs in non-stimulated cells and staining for perilipin is negative, the situation changes completely with the start of AIM stimulation. (For additional examples of small LD staining conspicuously arranged along rows of filaments, see e.g. TIP47 staining patterns obtained with PLC epithelial cells given in Fig. S5 by Heid et al., 2013 ). Nuclear staining was with DAPI (blue). Bars: 20 µm.