Ctb alexa 555

Retinoic acid was obtained from Sigma-Aldrich China (Shanghai, China; R2625). Rapamycin was purchased from MedChemExpress (Shanghai, China; HY-10219). CTB-Alexa 488 and CTB-Alexa 555 were bought from BrainVTA (Wuhan, Hubei, China). The following antibodies were used in western blot: anti-CD82 (ab66400 & ab135779; Abcam; 1:1000); anti-synaptophysin (ab14692; Abcam; 1:1000); anti-p70S6k (2708T; Cell Signaling Technology; 1:1000); anti-p-p70S6k(9234T;Cell Signaling Technology; 1:1000); anti-β-actin (sc-47778; Santa Cruz; 1:1000); anti-TRAF2 (ab126758; Abcam; 1:1000); anti-Raptor (20984-1-AP; Proteintech; 1:1000).The following antibodies were used in immunofluorescence: anti-CD82 (ab66400; Abcam; 1:200); anti-Tuj1 (801201; Biolegend; 1:1000); anti-Iba1(ab48004;Abcam;1:100); anti-GAFP(ab4674; Abcam;1:100); anti-mCherry (ab167453; Abcam; 1:100); anti-synaptophysin (ab14692; Abcam; 1:200); anti-pS6(Ser 240/244)(5364T; Cell Signaling Technology; 1:1000); anti-TRAF2 (ab126758; Abcam; 1:400); anti-Raptor (20984-1-AP; Proteintech; 1:1000); anti-Lamp1 (15665, Cell Signaling Technology; 1:500).
Anti-β-Amyloid (2450; Cell Signaling Technology; 1:100) was used in immunohistochemistry.

CTB-Alexa 555 (BrainVTA, Wuhan, Hubei, China) was intravitreally injected 48 h before sacrifice to trace regenerating RGC axons 14- or 28-days post-injury. After 4% PFA perfusion, mice eyes and optic nerves were microdissected and post-fixed for 3 h in 4% PFA. The nerves were dehydrated with 30% sucrose in PBS overnight at 4 °C and embedded in OCT Compound (Tissue-Tek, Sakura Finetek Inc, Tokyo, Japan) for cryosection. Optic nerves were cut longitudinally at a thickness of 10 µm and mounted onto slides. Alexa 555 was imaged using a fluorescent microscope (Olympus, Tokyo, Japan). Regeneration axons were quantified by counting the number of CTB-labeled fibers extended past every 500 µm division from the crush site. The total number of regeneration axons in each optic nerve was estimated using the equation elaborated in the literature [56 (link)].