Hp ol

The role of microglia in Aβ degradation was further examined in vivo. We stereotaxically injected 2 μl of 100 µM Rho-Aβ into the lateral ventricles of CX3CR1-GFP^+/− mice. GFP-positive microglia and rhodamine (Rho)-labeled plaques were imaged through a thinned skull preparation^{60 (link)}. The transgenic mice were anesthetized with 20% urethane filtered with a 0.22 µm filter (Millipore), and the skull was exposed with a midline scalp incision. An ~1-mm-diameter region of the skull over the somatosensory cortex was thinned with a high-speed drill and a microsurgical blade to a final thickness of ~40 μm. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate. A mode-locked laser (MaiTai HP-OL/Insight DS-OL, Spectra-Physics) was used for two-photon excitation and set to 920 nm for the imaging of GFP, 1120 nm for the imaging of Rho and 835 nm for the imaging of Methoxy-X04 using an in vivo two-photon imaging system (Olympus FVMPE-RS). Images were obtained by using a 1.05 numerical aperture, 25× water-immersion objectives and 2.0× digital zoom. The stack was typically obtained 150 μm below the pial surface^{61 (link)}.

RNA in situ hybridization and immunofluorescence images were acquired on a point scanning confocal microscope (model FV1000, Olympus). It was equipped with visible light (405, 442, 488, 515, and 647 nm) and pulsed infrared (MaiTai HP-OL, Spectra-Physics) lasers. It is equipped with one regular photon-multiplier tube (PMT), two spectral PMTs, two GaAsP detectors, two nondescanned detectors, and a motorized stage.
High-powered fields at 20×, 40×, and 60× objectives were used for the experiments to provide optimal resolution for the cells or features being identified: 60–100× for RNA-scope and BMAL1 quantification; and 20–40× for NG2-glia quantification. Regions of interest (ROIs) in naive animals were imaged from primary somatosensory cortex just lateral to the cingulum. For TBI experiments with cell quantification, two 40× objective high-power field were collected per slice, spanning a total area of 317 × 635 μm, centered around a peri-injury ROI at the lesion core as indicated by a combination of the following features: obvious surface deformation, increased background green fluorescence, overall cellular density, disorganized NG2 staining, and densest Ki67⁺ cell population.