Tissue preparation for fluorescence imaging

After the duration of the experiment, mice were transcardially perfused using cold PBS and paraformaldehyde (PFA) (4% in PBS pH 7.3). Tissues samples were removed, stored overnight in PFA at 4 °C, and re-stored in cold PBS. Brain was cut into 100 µm coronal sections using a vibratome (Leica VT1000E). Spinal columns (the cervical region), liver, kidney, lungs, and heart were embedded in Optimal Cutting Temperature compound and cut into 30 μm sections using a cryostat. In some experiments, the skin was removed from the head, then decalcified in 13% EDTA for 5 days, and cut using a cryostat. Sections were mounted on Superfrost Plus glass slides using ProLong Gold Antifade Mountant medium (ThermoFisher Scientific, Waltham, MA, USA) for fluorescence imaging (VS120 Virtual Slide Microscope, Olympus). Exposure and gain were fixed for all experimental groups based on pilot experiments. All fluorescence (SP-555 and OA-488) quantification was performed without enhancement of signals. The person performing the imaging and analysis were blinded to the experimental design and tracer used. This was finally decoded when the figures were prepared for publication.