Mouse anti neun antibody

Cytochalasin D (125 ng/µL in normal saline and 1% dimethylsulfoxide [DMSO]), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and anti-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Jasplakinolide (20 ng/µL in normal saline containing 2% DMSO) was obtained from Invitrogen (Carlsbad, CA, USA). Mouse anti-NeuN antibody was obtained from Abcam (Cambridge, UK). Rabbit anti-glial fibrillary acidic protein (GFAP) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), and rabbit anti-IBL-1 antibody was purchased from Wako Chemicals (Osaka, Japan). Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, were obtained from Thermo Fisher Scientific (Waltham, MA, USA). AffiniPure Goat Anti-Rabbit secondary antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).

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.

To evaluate the neuronal apoptosis, co-staining of neuronal apoptosis was conducted by NEUN staining and TUNEL staining. Slides were incubated with mouse anti-NEUN antibody (1:100, Abcam, UK) overnight at 4°C. TUNEL staining was conducted using Apoptosis Detection Kit (Roche, USA) as recommended by the manufacturers at 24 h after ICH. 4’,6-diamidino-2-phenylindole (DAPI) solution was used to label nuclei with blue fluorescence at 4°C. Apoptosis was observed under a fluorescence microscope (Zeiss, Germany). Three rats were examined per group and five visual fields were randomly selected from the hematoma area of the right basal ganglia area for each slide. The number of positive cells were counted using a cell-counter (Olympus, Japan). The positive neurons were identified by red cells (TUNEL) located in green neurons (NEUN) with a blue nucleus (DAPI). Data was expressed as ratio of positive neurons (%).