Ctb alexa 555

A subset of mice that were used for S1 somata imaging experiments were injected post hoc with a retrograde tracer, CTB-Alexa555 (5 µg µl⁻¹ in PBS, Invitrogen) in S2 localized by intrinsic signal imaging. The post hoc injection occurred shortly after conclusion of behavioral experiments. A small hole was drilled through the glass cranial window. One-hundred nanoliters of CTB-Alexa555 was injected (depth, 400–500 µm; rate, ~1 nl s⁻¹) through the hole via a glass pipette (30–50 µm). Injection site was sealed with dental cement. Seven to ten days after the injection, the labeled cells were examined under the two-photon microscope. To localize co-labeling, GCaMP6-labeled cells and CTB-Alexa555-labeled cells were excited at 940 nm or 1,020 nm, respectively (40–60 mW at specimen), respectively. Fluorescence emission was separated using a 568 nm dichroic (FF568-Di01-35.5x50.2, Semrock), passed through green (ET525/70m-2p, Chroma) and red (FF01-625/90-30-D, Semrock) channel filters before detection with two GaAsP photomultiplier tubes (Hamamatsu).

All experimental procedures were performed in compliance with animal protocols approved by the University Animal Care Committee at University of Toronto and IACUC at University of Miami. For all surgical procedures, young adult naked mole-rats (6-12 months old) were anesthetized with Avertin (30 mg/100 g; Sigma, St. Louis, MO). Adult C57BL/6J mice (IMSR catalog No. JAX:000664; RRID:IMSR_JAX:000664; 6-8 weeks old) were anesthetized with a ketamine and xylazine mixture. Buprenorphine (0.05 mg/kg, Bedford Laboratories, Bedford, MA) was administered as postoperative analgesic. For optic nerve crush injury, the left optic nerve was exposed intraorbitally and crushed with forceps for 10 seconds at 1 mm behind the optic disc. For intravitreal injection, a glass micropipette was inserted into the peripheral retina, just behind the ora serrata, and was deliberately angled to avoid damage to the lens. Two days before dissection, approximately 0.5 μl (for naked mole-rat) or 2-3 μl (for mouse) of cholera toxin β subunit (CTB)-Alexa 555 (2 μg/μl, Invitrogen, Carlsbad, CA) was injected into the vitreous with a Hamilton syringe (Hamilton) to label regenerating RGC axons anterogradely.