Anti glial fibrillary acidic protein antibody

ELISA, western blot and immunohistochemistry assays were performed to examine the expression of human BDNF in the brains of P301L mice after intraventricular injection of AAV-BDNF. Fresh left hemisphere was weighed and homogenized in lysate buffer. The homogenates were centrifuged at 10  000 g for 10 min at 4 °C, and the resultant supernatant was collected and subjected to ELISA assays according to the manufacturer's protocols (human BDNF ELISA kit, Abbexa). In addition, the levels of total BDNF including endogenous mouse BDNF and human BDNF in brain homogenates were also detected by western blot analysis using anti-BDNF antibody (Abcam, ab72439, detecting both mouse and human BDNF). Coronal sections from the right hemisphere were immunostained for microglia (anti-CD45 antibody, Millipore, Bedford, MA, USA), astrocyte (anti-glial fibrillary acidic protein antibody, Millipore), neurons (anti-NeuN antibody, Abcam), GFP (anti-GFP antibody, Abcam) and human BDNF (anti-human BDNF antibody, R&D systems, Minneapolis, MN, USA) to identify localization and levels of human BDNF expression in P301L mouse brains after AAV-BDNF injection.

Cells were fixed in 4% PFA. For immunostaining, brain sections were randomly chosen based on the anatomical location from different serial collecting wells for an individual rat. Each collecting well contained approximately 6–8 serial sections from anterior to posterior at a spacing of every 300 μm. Blocking solution for fixed cells and brain sections included 10% normal donkey serum (Jackson Immunoresearch) in PBS surplus with 0.4% Triton X-100. Primary antibodies (Tyrosine Hydroxylase: millipore; DDC Antibody: Novus Biologicals; Anti-GCH1 Antibody: abcam; Mouse anti-human nuclei monoclonal antibody: Millipore; Ki-67 Monoclonal Antibody: Thermo Fisher Scientific; Anti Iba1: Wako; Anti-CD45: Bioss; Anti-Glial Fibrillary Acidic Protein Antibody: millipore) were diluted in PBS containing 2.5% normal donkey serum and 0.4% Triton X-100 and incubated overnight at 4 °C according to manufacturer recommendations. Secondary antibodies conjugated to Alexa488, Alexa555, or Alexa647 (Molecular Probes) diluted in PBS containing 5% normal donkey serum were incubated for 2 h at room temperature. Nuclear counterstain was visualized with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher). After PBS wash, stained sections were mounted on adhesion microscope glass slides (MXB Biotechnologies).