Mouse anti gfap antibody

Mice were anesthetized with 2% pentobarbital sodium, transcardially perfused with 0.9% saline, and fixed with 4% paraformaldehyde. Then, the brain tissues were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. For immunohistochemical detection of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) expression, brain tissue is prepared into 5-μm coronal sections. First, sections were deparaffinized by xylene and gradient alcohol, and after heat-mediated antigen retrieval with citrate buffer or ethylene diamine tetraacetic acid, sections were stained according to the steps of the immunohistochemistry kit (MX Biotechnologies Co., Ltd, Fuzhou, China). Brain sections were then rinsed briefly in phosphate-buffered saline, treated with 1% hydrogen peroxide for 10 min, and blocked for 10 min. Next, the sections were incubated with a mouse anti-NeuN antibody (1:1000; Abcam), mouse anti-GFAP antibody (1:1000; Proteintech), and rabbit anti-MBP (1:200; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. They were incubated with secondary antibody and streptomyces antibiotic protein-peroxidase on the second day. Diaminobenzidine chromogenic agent was used to develop color. And ImageJ Version 1.8.0 (NIH, New York, NY, USA) was used to analyze the average optical density values for quantification.