Immunofluorescence Labeling of Neural Markers

After blocking (×1 PBS, 2 % normal goat serum, and 0.1 % Triton X-100) for 1 h, the sections were incubated overnight at 4 °C with a mixture of any two of the following primary antibodies: anti-CXCL5 (1:50; abcam), anti-ED1 (1:100; Chemicon, Temecula, CA), anti-O4 IgM (1:100; Chemicon), anti-GFAP (1:200; Chemicon), anti-neuronal nuclear antigen (NeuN) (1:200; Millipore Bioscience Research Reagents), and anti-RECA (1:100; abcam). The sections were washed three times with 0.1 M PBS and incubated with Alexa Fluor 594 anti-mouse IgG/IgM or Alexa Fluor 488 anti-rabbit IgG (1:400; Invitrogen) for 1 h at room temperature. The fluorescence signals were detected, and the results were recorded using a microscope (E400; Nikon Instech) at excitation–emission wavelengths of 596–615 nm (Alexa Fluor 594, red) and 470–505 nm (Alexa Fluor 488, green) [18 (link), 23 (link)].