Axiocam 702 mono

Male AgRP^Cre/+;Rosa26^tdTom and AgRP^Agpat5KO;Rosa26^tdTom (12 weeks old, ad libitum fed) were subjected to a trans-cardiac perfusion of fresh cold fixative solution (1% glutaraldehyde/2% cold PFA in 0.1 M phosphate buffer, pH 7.4). Brains were isolated and kept overnight in the fixative solution. After two washes with PB solution, 70 μm coronal sections containing ARC were prepared using a vibratome (VT1000S, Leica) and tdTomato-positive fluorescent cells were imaged using a ZEISS Axio Imager.M2 microscope, equipped with ApoTome.2 and a Camera Axiocam 702 mono using the AxioVision software (Zeiss, Oberkochen, Germany). Sections were osmicated (1.5% osmium tetroxide) for 1 h followed by 1 h incubation in 1% (wt/vol) tannic acid in 0.1 M PB buffer. Samples were then dehydrated with ethanol solutions (1% uranyl acetate in 70% ethanol for 30 min) and flat-embedded in Epon-araldite. Trimmed blocks were used to cut ultrathin sections that were examined using a Philips CM-10 electron microscope^{41 (link)}. Electron microscopy images were cross-referenced with immunofluorescent images of tdTomato-positive AgRP neurons to identify the cell population of interest. Mitochondrial morphology in AgRP neurons was analyzed using ImageJ software, quantifying mitochondrial area and perimeter in tdTomato-positive AgRP neurons^{42 (link)}.

For live imaging, cells were washed and resuspended in 10 mM Tris pH 7.4 and placed directly on glass slides, where some cells become immobilized by the pressure of the cover slip. Alternatively, cold CyGel (Abcam) was buffered with 1 M Tris, pH 7.4 (100:1, for a final concentration of 10mM), and this was mixed 1:1 with cells on coverslips, which were then gently pressed onto slides at room temperature. Images were captured with an Axiocam 702 mono using the Zeiss Axio Observer 7 system, with a 100X objective (Alpha Plan-Apo 100x/1.46 oil DIC M27).