Lsm 880 airyscan two photon confocal microscope

BAT SVF cells were seeded in 24-well plates with high performance #1.5 cover glass bottoms (Cellvis P24-1.5H-N), grown to confluence and then treated with brown adipocyte differentiation cocktail as previously described^{20 (link)}. Four days after the initiation of differentiation, the cells were transduced with scrambled, TMEM135 shRNA, pLJM1-EGFP or TMEM135 overexpression lentivirus in the differentiation medium, as indicated. After 48 h, the viral medium was removed and fresh differentiation medium was added. On day 9 of differentiation, the medium was removed and the cells were washed with pre-warmed PBS before addition of phenol red-free DMEM containing tetramethylrhodamine, ethyl ester (TMRE) to label mitochondrial networks, and the cells were imaged using a Zeiss LSM 880 Airyscan Two-Photon Confocal Microscope. Mitochondrial morphology was quantified by visual inspection, and cells were divided into three groups, exhibiting fragmented, fused, or intermediate (partially fragmented) mitochondria, as previously described.

Mice were perfused with Heparin (Alfa Aesar) solution followed by 4% PFA (Microscopy Sciences) before harvesting tissues for fluorescence imaging. Bones were fixed in 4% PFA overnight. Zeiss LSM 880 Airyscan Two-Photon Confocal Microscope was used to image PyMT-Bo1 metastatic lesions in the intact bones. To image DiD + cells in the bone, harvested bones were decalcified in 14% EDTA pH = 7.2 for 3 days before cryo-sectioning (10 µm sections). Samples were mounted with Fluoroshield containing DAPI (Sigma) to preserve the DiD fluorescence and imaged by Nikon Eclipse Ti-E microscope.