Mouse monoclonal anti glial fibrillary acidic protein

The brains were perfused with 4% paraformaldehyde in PBS (pH 7.4) and frozen. Immunohistochemistry was performed on 30 µm sections using rabbit polyclonal anti-mouse Ngb (Sigma 1:100), mouse monoclonal anti-NeuN (Chemicon; 1:200), rat monoclonal anti-PDGFRβ (Abcam; 1:100), mouse monoclonal anti-αSMA (Abcam; 1:100), mouse monoclonal anti-glial fibrillary acidic protein (Sigma; 1:200), as primary antibodies, and Alexa Fluor 488-conjugated goat anti-rat IgG (Abcam; 1:500), Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen; 1:500), and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen; 1:500) as secondary antibodies. Controls included omitting primary or secondary antibodies. Fluorescence signals were detected using a Zeiss LSM 800 confocal laser scanning microscope at excitation/emission wavelengths of 495/519 (Alexa Fluor 488), 556/573 (Alexa Fluor 546), 590/617 (Alexa Fluor 594), and 358/461 (DAPI) nm. In order to quantify Ngb expressed around blood vessels, we measured Ngb signals within 2 mm of all blood vessels using 512×512-pixel figures by image J.

For the immunohistochemical analysis of rat spinal cords, we used mouse monoclonal anti–glial fibrillary acidic protein (Sigma-Aldrich, St. Louis, MO, USA) for astrocytes and rabbit anti–ionized calcium-binding protein-1 (Iba-1) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for ramified microglia and macrophages.
To detect GSK-3β, monoclonal rabbit anti-phospho-GSK-3β (Ser⁹) (p-GSK-3β) and monoclonal rabbit anti-GSK-3β antibodies were used (Cell Signaling Technology, Beverly, MA, USA). Monoclonal rabbit anti-β-catenin (Ser⁶⁷⁵) and PhosphoPlus Akt (Ser⁴⁷³) antibody kits were purchased from Cell Signaling Technology. Rabbit polyclonal anti–vascular cell adhesion molecule-1 (VCAM-1) antibody (H-276, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to assess the severity of inflammation. A mouse monoclonal anti-β-actin (Sigma-Aldrich) antibody was also used.