Gnl 10

A Thy1-GFP line M transgenic mouse (the Jackson laboratory, stock 007788) was used for preparation of brain slices. After being deeply anesthetized with isoflurane (Piramal), a standard transcardial perfusion was performed first with phosphate-buffered saline (PBS; Invitrogen) followed by 4% paraformaldehyde (PFA; Electron Microscopy Sciences). The mouse brain was collected and immersed in 2% PFA and 15% sucrose in PBS solution overnight at 4°C. The immersion solution was then replaced with 30% sucrose in PBS, and the brain was stored at 4°C. After 24 hours, the whole mouse brain was cut to 100-μm-thick slices on a microtome (Thermo Scientific, Microm HM430). Brain slices were immersed in PBS and then placed on microscope glass slides, and allowed to dry for 45 min. Cover glass (Fisherbrand, No. 1.5, 0.16 to 0.19 mm thick) with mounting medium (Vectashield Hardset Antifade mounting medium, H-1400) was then placed on top of the glass slides with brain slices. Slices were ready for imaging after the mounting medium completely hardened. During imaging, brain slice samples were placed on a 2D goniometer platform (Thorlabs, GNL 10).

Five microliters of stock solution of 0.1-μm-diameter fluorescent beads (ThermoFisher Scientific FluoSpheres Carboxylate-Modified Microspheres, yellow-green fluorescent 505/515) was mixed with 4 ml of 1% aqueous agarose solution. This mixture was then poured onto a glass-bottomed petri dish (MatTek No. 1.5 coverslip, 0.16 to 0.19 mm thick) and was allowed to solidify at room temperature. Beads were imaged through the No. 1.5 coverslip. Tilting of the 3D beads sample was controlled by a 2D goniometer platform (Thorlabs, GNL 10).