Polyclonal rabbit anti gap43 antibodies

Mice were perfused, optic nerves and eyes were dissected and post fixed overnight in 4% paraformaldehyde at 4°C. Nerves were transferred to a 30% sucrose/PBS solution and maintained at 4°C for at least 2 h and up to 2 wk. Optic nerves were imbedded in Tissue-Tek OCT Compound (Sakura Fintek USA, Inc.) and stored at −80 °C. Longitudinal sections (10 μm thick) were cut with a cryostat, mounted on Superfrost Plus microscope slides (Fisher Scientific), and stained with polyclonal rabbit anti-GAP43 antibodies (Abcam). Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (Invitrogen) was used for fluorescent labeling. Images were acquired using an Olympus IX71 inverted microscope with Olympus Cellsens Dimension 2.0 software, attached to an Olympus DP72 digital camera. Regenerating GAP43+ axons were counted at each 0.2-mm interval past the injury site, up to 1.6 mm, using a superimposed grid (3–5 sections/ nerve). The number of labeled axons per section was normalized to the width of the section and converted to the total number of regenerating axons per optic nerve, as described previously⁴³ .