Rabbit anti vegf a antibody

Frozen 20-um-thick brain sections were cut using a freezing microtome (CRYOSTAR NX50, Thermo Fisher Scientific, USA). The brain sections were then blocked by 50% goat serum (Solarbio, Beijing, China) and incubated at 4 °C for 12 h with specific primary antibodies: i.e., rabbit anti-NeuN antibody (1:200, Abcam, UK), rabbit anti-CD34 antibody (1:300, Abcam, UK), and rabbit anti-VEGF-A antibody (1:100, Abcam, UK). After washing three times in PBS, the NeuN and CD34 sections were incubated with the secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor 488, 1:500, Abcam, UK) at 37 °C for 2 h, and the VEGF-A sections were incubated with the secondary antibody goat anti-rabbit IgG H&L (Cy3, 1:500, Abcam, UK) at 37 °C for 2 h. The sections were then washed with 0.01 M PBS (Solarbio, Beijing, China) and labeled using DAPI (Fluoroshield Mounting Medium with DAPI, Abcam, UK). Images were obtained using a fluorescence microscope (Ci-L, Nikon, Japan).
Positive NeuN and VEGF-A expression was visualized under a microscope (20× objective), and five images of each region were taken for counting fluorescent area using ImageJ software (http://rsb.info.nih.gov/ij/). In addition, the three regions with the highest CD34-positive density were selected under a microscope (4× objective), and five images of each region were taken for counting (20× objective).