Goat anti ang 2

To identify where Ang-2 and MMP-9-positive signals were expressed, ANG-2 and MMP-9 was co-stained with NeuN (neuronal marker), glial fibrillary acidic protein (GFAP, astrocyte marker), CD11b (microglial marker), or Lectin (blood vessel marker). In addition, a single immunofluorescent staining for VEGF was conducted to observe its distribution in the hippocampus. Three sections per animal were washed in PBS with 0.15% Triton X-100 and then incubated in blocking serum, 5% normal horse serum in 0.15% triton with PBS. The sections were then incubated in primary antibody cocktail for 22 hours at room temperature. For primary antibody, mouse anti-NeuN (1:1000, Millipore), mouse anti-GFAP (1:500, BD bioscience, San Jose, CA), mouse anti-CD11b (1:500, Bio-Rad, Hercules, CA), rabbit anti-MMP-9 (1:100, Millipore), goat anti-Ang-2 (1:200, Santa Cruz), and FITC-conjugated Tomato-Lectin antibody (1:500, Vector, Burlingame, CA), mouse anti-VEGF antibody (1:500, Santa Cruz) were used. Sections were washed in PBS with 0.15% Triton X-100 and incubated in the Alexa® fluore conjugated secondary antibody cocktail solution (1:200, Invitrogen, Waltham, MA) for 2 hours at room temperature. Stained sections were mounted on resin-coated slides and dried for 30 min. Anti-fade solution (Prolong gold, Invitrogen) was used to maintain the fluorescence.