Standard fluorescent microscope

Mounted sections were washed three times with PBS followed by blocking in 5% normal goat serum (NGS) and 0.1% bovine serum albumin (BSA) in PBS. 0.1% Triton X-100 was added to the blocking buffer depending on the antigen used. Following blocking, sections were incubated in primary antibody diluted in blocking buffer overnight at 4° C. Primary antibodies used were mouse anti-NeuN (1:100, Chemicon), anti-GFAP (1:1,000, Dako), mouse anti-ED1 (Chemicon, 1:100) rabbit anti-5-HT (1:500, Immunostar, Hudson, WI), and anti-Neurofilment (1:500, Sigma-Aldrich). For in vivo PTPσ staining, sagittal 20µM sections encompassing both rostral and caudal to the lesion were probed with anti-PTPσ antibody (1:500, Abnova). The next day, the sections were washed extensively with PBS and incubated in the appropriate secondary antibody or avidin substrate conjugated to Alexafluor 488, 594, or 633 (1:500, Molecular Probes) overnight. After extensive washing, the sections were stained with DAPI (1:1,500 in PBS, Sigma-Aldrich), washed, coverslipped, and viewed with a confocal microscope (Zeiss, Germany). Pixel intensity was measured on images taken on a standard fluorescent microscope (Leica, Germany) with a uniform exposure setting and analyzed using ImageJ.