Rabbit anti nestin polyclonal antibody

Wild-type C57BL/6 mice were obtained from Jackson Labs, Bar Harbor, ME and kept under pathogen free conditions (Department of Comparative Medicine, Washington University, St. Louis, MO). Experiments with mice were performed under approved guidelines as outlined by the Washington University School of Medicine Animal Studies Committee. Purified mouse monoclonal anti-LCMV-nucleoprotein antibody (1.1.3) from hybridoma supernatant was provided by Dr. J. Carlos de la Torre (The Scripps Institute, La Jolla, CA). Sheep anti-BrdU, chicken polyclonal anti-beta III tubulin, and goat polyclonal anti-IBA1 antibodies were purchased from Abcam. Rabbit anti-doublecortin (DCX) polyclonal antibody was purchased from Cell Signaling. Rabbit anti-nestin polyclonal antibody was purchased from Millipore. Fluorescently-conjugated antibodies against CD45.2 (fluoroscein isothiocyanate, FITC) was purchased from BD Pharmingen. Rat monoclonal anti-GFAP antibody and fluorescently-conjugated secondary antibodies were purchased from Invitrogen. Normal goat and donkey sera and isotype controls were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

A rat glioma model was established to test the in vivo effect of NA. The C6 glioma cells were orthotopically injected to the right striatum of Sprague-Dawley (SD) rat brains through a pre-settled stainless steel tube. NA and control (PBS) were also injected through the tube. When the treatment was completed, the rats were sacrificed by anesthesia overdose and their brain tissues removed and embedded immediately in optimal cutting temperature compound. The brain tissues were subsequently cut into slices (7-μm thick) with a Leica CM1850 cryostat microtome. Brain slices were fixed with 95% ethanol and stained with hematoxylin and eosin (both were from Sigma). For immunohistochemistry, brain slices were fixed with 4% paraformaldehyde for 30 min, followed by blocking with 5% BSA for 1 hr at room temperature. The slices were then incubated with rabbit anti-Nestin polyclonal antibody (Millipore) at 4 °C overnight, washed, incubated again with phycoerythrin-conjugated secondary antibody (Santa Cruz Biotechnology) for 45 min at room temperature, and stained with DAPI to label nuclei. After being sealed with neutral mounting medium (Jiangyuan, Jiangsu, China), the slices were observed under an Olympus upright microscope.

Brain tissue sections were de-waxed and rehydrated, followed by microwave antigen retrieval procedure. After blocking non-specific binding using 10% normal goat serum, the following primary antibodies were added and incubated at 4°C overnight: mouse anti-BrdU monoclonal antibody (1:500, Cell Signaling Technology, Danvers, MA), rabbit anti-nestin polyclonal antibody (1:500, Sigma-Aldrich, St. Louis, MO) and rabbit anti-doublecortin polyclonal antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA). Next the slides were incubated with respective FITC and TEXAS red-conjugated secondary Abs followed by washing and mounting with DAPI-containing medium (Vectashield H-1200) for fluorescent microscopy.