Uplfln 20x

On the day of microscopy imaging, the frozen brains were transferred to a −20°C cryostat microtome (CM1950, Leica Biosystems) and coronally cut to slices of different thicknesses. The sections were placed on standard microscope slides within a closed slide box for air-drying at room temperature for 5–60 min. Three different slice section thicknesses of 12, 20 or 50 μm were tested for the control brains, to optimize the number of clearly visible blood vessels with a good signal and reduced blurriness that can be a byproduct of out of focus planes (Figure 1). The optimal thickness of 20 μm was selected for the treated brains and the PFA preserved brains. The brain slides were imaged within 1 h of sectioning to avoid further EB perfusion.⁴² (link) Images were acquired with a motorized upright fluorescence microscope (BX63, Olympus life science, Waltham, Massachusetts, USA) with an ×20 objective (UPLFLN 20X, Olympus life science). This objective had a focal depth of 1.1 μm. The excitation wavelengths and exposure times were 508 nm and 1150 msec for FITC-Dextran, and 615 nm and 115 msec for EB. All the imaging parameters were consistent for all sections. The final images were 1920 × 1200 pixels in size, with a pixel size of 0.29 μm in the imaging plane.