Dumont 5

Hindlimbs from WT and mutant mice were skinned and fixed for 24–30 h in 10% neutral buffered-formalin, rinsed twice, and stored in 70% ethanol at 4 °C. Samples were anonymized and randomly assigned to batches for rapid-throughput analysis. Prior to analysis, limbs were rehydrated in PBS + 0.02% sodium azide for >24 h. Soft tissue was removed and the knee joint was disarticulated under a Leica MZ9 dissecting microscope (Leica Microsystems, UK) with the aid of fine forceps (Dumont #5, Cat#11252-20; Fine Science Tools, Germany) and 5 mm spring scissors (Vannas Tubingen, Cat#15003-08, Fine Science Tools, Germany).

Intravitreal injections of AAV2 viruses, ONC, and RGC axon labeling were performed as previously described (Park et al., 2008 (link)). Animals were first anesthetized, 1.5 μl of AAV2 virus or Alexa Fluor 555-conjugated CTB (1 μg/μl, Thermo Fisher Scientific, Waltham, MA, USA) was then injected into their right vitreous humor with a Nanoliter syringe. CTB-555 injection was performed 2 days before the mouse was sacrificed to trace regenerating RGC axons. The optic nerve was exposed intraorbitally and crushed with forceps (Dumont #5, Fine Science Tools) for 5 s approximately 1 mm behind the optic disc. The mice were transcardially perfused with 4% paraformaldehyde (PFA). Dissected right optic nerve and bilateral retinas were post-fixed in 4% PFA overnight. pCMV-GFP and pCMV-Cre were from Vigene Bioscience. The titers of AAV2-CMV-Cre and AAV2-CMV-GFP were 1.62 × 10¹³ vg/ml and 3.2 × 10¹³ vg/ml, respectively.

Mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine/g+0.08 mg ketamine/g). For each AAV intravitreal injection, a micropipette was inserted into the peripheral retina of 3 week-old mice just behind the ora serrata, and advanced into the vitreous chamber so as to avoid damage to the lens. Approximately 2 µl of the vitreous was removed before injection of 2 µl AAV into the vitreous chamber. ON crush was performed 2 weeks following AAV injection: the ON was exposed intraorbitally and crushed with a jeweler’s forceps (Dumont #5; Fine Science Tools, Forster City, California) for 5 s approximately 0.5 mm behind the eyeball. Care was taken not to damage the underlying ophthalmic artery. Eye ointment containing neomycin (Akorn, Somerset, New Jersey) was applied to protect the cornea after surgery.

Mice were anesthetized by xylazine and ketamine based on their body weight (0.01 mg xylazine/g + 0.08 mg ketamine/g). Optic nerves of both sides were sequentially exposed intraorbitally and crushed with a jeweler’s forceps (Dumont #5; Fine Science Tools) for 2 s approximately 0.5 mm behind the eyeball. Care was taken not to damage the underlying ophthalmic artery. Erythromycin Eye Ointment was applied to protect the cornea after surgery.

Experimental procedures were performed in compliance with animal protocols approved by the Animal and Plant Care Facility at the Hong Kong University of Science and Technology. C57BL/6 mice of 5–6 weeks of age were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) and received Meloxicam (1 mg/kg) as analgesia after the surgery. AAV-vectors (serotype 2/2, titer 1 × 10¹² vg/ml, 2 μl injection volume) expressing either Cpeb1-HA or Gfp under the neuron-specific human synapsin promoter (hSyn) were injected into the left vitreous bodies of 12 mice with a Hamilton microsyringe. Five weeks after vector injection, the optic nerve was gently exposed intraorbitally and crushed with jeweler's forceps (Dumont #5; Fine Science Tools) around 1 mm behind the optic disk. Mice were kept for 2 weeks after injury before tracing. To visualize RGC axons in the optic nerve, 1.5 μl cholera toxin β subunit conjugated with Alexa555 (CTB555, 2 μg/μl, Invitrogen) were injected into the vitreous bodies. Two days after the CTB injection, animals were sacrificed by transcardial perfusion for histology examination. In each mouse, the completeness of optic nerve crush was verified by showing that anterograde tracing did not reach the superior colliculi.