Clone hm 2

Brain sections were deparaffinized in xylene and rehydrated in graded ethanol, and antigen retrieval was performed by heating the sections in 10 mM boiling sodium citrate buffer (pH 6.0) for 10 min and allowing them to cool for 30 min. Non-specific binding was blocked by incubating with 4% donkey or goat serum in PBS for 30 min, and endogenous peroxidase activity was blocked with 3% H₂O₂. The primary antibodies were as follows and were diluted in PBS and incubated with the sections overnight at 4 °C: monoclonal mouse anti-MAP2 (1:1,000 dilution, clone HM-2, Sigma, M4403), monoclonal mouse anti-MBP (1:500 dilution, clone SMI94, BioLegend, 836,504), polyclonal rabbit anti-cleaved caspase-3 (1:200 dilution, Asp175, Cell Signaling, 9661), and monoclonal rabbit anti-AIF (1:500 dilution, E20, Abcam, ab32516) antibodies. After the primary antibody incubation, the appropriate biotinylated secondary antibodies (1:200 dilutions, all from Vector Laboratories, Burlingame, CA, USA) were added to each section for 60 min at room temperature. The sections were visualized with a Vectastain Elite ABC HRP Kit (Vector Laboratories, PK-6100) and 0.5 mg/mL 3,3′-diaminobenzidine enhanced with ammonium nickel sulfate, β-D glucose, ammonium chloride, and β-glucose oxidase. After dehydrating with graded ethanol and xylene, the sections were mounted on coverslips with Vector mounting medium.

Due to the lower capability for dendritic outgrowth, compared with axons, the central region of 2 mm × 20-mm silicone barrier area normally contained axons only. Microtubule-associated protein 2 (MAP2, a specific marker for dendrites and neuronal somata) and microtubule-associated protein Tau (a specific marker for axons and neuronal somata) were used to identify neurites in the axon-only area. Cells were labeled with either monoclonal mouse anti-MAP2 antibody (1:2,000; clone HM-2, Sigma-Aldrich, St. Louis, MO, United States) or Tau (1:1,000; clone Tau46, Sigma-Aldrich, St. Louis, MO, United States), followed by Alexa Fluor 594 goat anti-mouse IgG1 (1:100 Molecular Probes, Thermo Fisher Scientific, Rochester, NY, United States). Detailed methods are described below in the section relating to immunocytochemistry. To test the purity of our neuronal cell cultures, cultured cells were double labeled with polyclonal rabbit anti-β-tubulin antibody (1:500, Cell Signaling, Danvers, MA, United States) and a neuronal cell maker (either MAP2 or Tau). Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG were used as corresponding secondary antibodies. A high ratio of MAP2 to Tau-positive cells compared to β-tubulin III-positive cells demonstrated the purity of the cultured neurons.