Glial fibrillary acidic protein (gfap)

IHC of ED1 (CD68, a marker of macrophage/microglia) in the ONs and IHC of GFAP in the retina using monoclonal antibody (ED1, 1:50; AbD Serotec, Oxford, UK) or polyclonal antibody (GFAP, 1:200; Cambridge, MA, USA) was performed. Briefly, the frozen longitudinal sections of ONs and retina were fixed with acetone at -20°C for 30min and blocked with 5% fetal bovine serum (FBS) containing 1% bovine serum albumin (BSA) for 15 min. The anti-ED1 antibody (1:50 AbD Serotec, Oxford, UK) was applied and incubated at 4°C overnight. The secondary antibody conjugated with fluorescein isothiocyanate (FITC, 1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was applied at room temperature for 1h. Counterstaining was performed using DAPI (1:1000, Sigma, St. Louis, MO, USA). For comparison, ED1 positive cells were counted in six HPF at the lesion site of ON (n = 6 in each group).

Triple staining was performed to identify the phenotype of the new cells (Fig 1).
Sections were processed following the immunohistochemistry protocol described above. Samples were incubated in primary antibody rat anti BrdU (1:800, AbD Serotec) for at least16 h at 4°C followed by incubation for 2 h with the secondary antibody rat IgG (1:500, Vector). After incubation, the samples were placed in ABC and revealed with tyramide signal amplification (TSA) plus coumarin system (1:100, PerkinElmer) according to vendor’s instructions. Sections were rinsed and incubated in mouse monoclonal anti- neuronal nuclei antibody (NeuN) to identify mature neurons (1:250, Millipore) for 20 h at 4°C and incubated in mouse IgG (1:300, Vector). Later, samples were incubated in ABC complex and revealed using TSA plus cyanine 3 (CY3) system (1:100). Sections were rinsed and incubated with primary antibody rabbit monoclonal anti- glial fibrillary acidic protein to identify glial cells (GFAP, 1:100, AbD Serotec) and incubated with secondary anti-rabbit IgG Alexa Fluor 488 (Life science, 1:1250). Finally, brain sections were mounted on slides and cover slipped using aqua poly/mount (Polysciences).