Cd34 antibody

Sections were processed for immunofluorescence staining with CD34 antibody to label microvessels, NeuN antibody to label neurons, Iba1 and GFAP antibodies to label microglia and astrocytes. Briefly, sections were boiled to recovered antigen in 0.01 mol/L citrate buffer (pH 6.0) at 96°C to 98°C for 15 minutes. Nonspecific binding sections were blocked with 5% normal goat serum or donkey serum in PBS at room temperature for 2 hours. Sections were then incubated with CD34 antibody (from rabbit, 1 : 200, Boster, Wu Han, China) and NeuN antibody (from rabbit, 1 : 1,000, Abcam, British) separately; Iba1 antibody and GFAP antibody (from goat or from mouse, 1 : 1,000, Abcam, Cambridge, MA, USA) for double-labelling overnight at 4°C. After PBS washes, the sections were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit lgG or donkey anti-rabbit, or donkey anti-mouse and Alexa Fluor 546-conjugated donkey anti-goat, Jackson Immunoresearch, West Grove, PA, USA) at 37°C for 2 hours. The concentration of all secondary antibodies was 1 : 1,000, then washed in PBS three times (5 minutes each) and covered with anti-quenching fluorescence mounting medium. The sections of cerebellum were performed a regular HE staining process.