Rc 27l bath

Two-photon (2p) imaging of hypothalamic slices was performed using a multiphoton microscope built on an Olympus BX51 upright microscope frame, fitted with a motorized Z-Deck stage (Prior Scientific, Cambridge, UK) and Nikon 25×, water dipping objective (CFI75 Apo L, NA = 1.1, WD = 2.0 mm). 2p excitation was generated by a tunable femtosecond laser (Chameleon Ultra-II or Vision-II, Coherent, Santa Clara, CA, USA) set to 920 nm to excite EGFP and TdTomato as described previously (Othy et al., 2020 (link)). Fluorescence emission was split by 484 nm and 538 nm dichroic mirrors into three non-descanned PMT detectors (R3896, Hamamatsu Photonics, Bridgewater, NJ, USA) to detect second-harmonic signal generated from collagen in blue; EGFP signal in green; and TdTomato signal in red. Supported brain slices were anchored in a RC-27L bath (Warner Instruments, Holliston, MA, USA) assembled into a custom-built heated imaging chamber maintained at 37° ± 0.5°C using continuous perfusion of oxygenated imaging media. 3D image stacks of x = 250 μm, y = 250 μm, and z = 56 μm (XYZ voxel size 0.244 μm × 0.244 μm × 4 μm) were acquired every 37 s interval using image acquisition software Slidebook (Intelligent Imaging Innovations, Denver, CO, USA), up to 50 min to create a 4D data set. Imaris version 9.2.1 or later (Bitplane) was used for rendering and image processing for figures.