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. 2021 Feb 11;62:100048. doi: 10.1016/j.jlr.2021.100048

Fig. 5.

Fig. 5

Ceroid-like structures and multilamellar bodies accumulate in the adrenal cortex-medulla border in Plin2−/− mice. Adrenals from 30-week-old ad libitum fed female Plin2+/+ and Plin2−/− mice were fixed and sectioned. Tissue sections were imaged with whole section scanning, confocal microscopy, or transmission electron microscopy (TEM). A: Whole section scans at the center planes of adrenals taken under bright field (BF) visualize accumulation of light brown-yellow aggregates in the basal adrenal cortex (close to medulla) in Plin2−/− adrenals. The same sections were subsequently stained with Hoechst 33342 to visualize nuclei. Nuclei signals were exited at 330–375 nm and detected at 430–470 nm, whereas the autofluorescent aggregates (orange) were exited at 545–557 nm and visualized at 578–640 nm (FL). Scale bars are 500 μm. B: Confocal imaging of the above Hoechst 33342-stained sections (40× objective from the square marked in images in A). The granule-like aggregates in Plin2−/− adrenals excited with a 405 nm laser have broad emission spectra (415–615 nm). Scale bars are 100 μm. C: Adrenal sections from Plin2+/+ and Plin2−/− mice were stained with Hoechst 33342 to visualize nuclei (blue), Bodipy 493/503 to visualize lipid droplet (green), and CF568-Phalloidin to visualize F-actin in plasma membranes (red). High-resolution confocal imaging (63× objective, marked with a box) reveals multiple nuclei and vesicles (white asterisks) and deformed cortical cells containing lipid droplets (white arrows) relatively restricted to Plin2−/− mice (scale bars 100 or 20 μm). D: Durcupan embedded ultra-thin sections from adrenals visualized with transmission electron microscopy. Examination of areas containing aggregates (encircled with yellow dotted lines) with higher resolution (red squared boxes indicate zoomed areas) reveals the presence of nonstructured lipid-like material (black asterisks) surrounded by multilamellar bodies (black arrow heads). Scale bars in images from left to right are 20 μm, 1 μm, and 300 nm, respectively. BF, bright field; Em, emission wavelength; Exc, excitation wavelength; FL, fluorescence.