Anterograde Transport of RGC Axons to SC

We determined the fidelity of anterograde transport of RGC axons to their target neurons in the SC by measuring the transfer of cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (CTB; Molecular Probes, Eugene, OR, USA). We intravitreally injected 1.5 μl of 1 μg/μl solution of CTB using a Hamilton syringe (7643-01; Hamilton, Reno, NV, USA) in anaesthetized mice (2.5% isoflurane). Two days after CTB injection, we transcardially perfused mice as described earlier.^{15–19 (link),20 (link),1–5 (link)} Following perfusion, we dissected out brains and cryoprotected in 20% sucrose solution. We then obtained coronal midbrain sections (50 µm) using a freezing sliding microtome (SM2000R; Leica Biosystems, Buffalo Grove, IL, USA). We imaged CTB fluorescence in alternating sections of the SC using a Nikon Ti Eclipse microscope. We quantified CTB signal intensity using a custom ImagePro (ImagePro 7; Media Cybernetics, Rockville, MD, USA) macro as described previously.^{15 (link)}