Robo4 anti mouse polyclonal antibody

Tissue sections were rinsed in phosphate-buffered saline (PBS) three times and then permeabilized with 0.5% Triton X-100 (Beyotime, Jiangsu, China) in PBS for 15 min at room temperature. Slides were blocked with 1% bovine serum albumin in PBS for 30 min at 37 °C. Subsequently, tissues were treated with 1:150 CD34 anti-mouse polyclonal antibody (Affinity, USA) plus 1:150 VEGF anti-rabbit monoclonal antibody (Affinity, USA), 1:150 CD34 anti-rabbit polyclonal antibody (Abcam, USA) plus 1:50 Robo4 anti-mouse polyclonal antibody (Santa Cruz Biotechnology, USA), and 1:150 VEGF anti-rabbit monoclonal antibody plus 1:50 Robo4 anti-mouse polyclonal antibody, respectively, at 4 °C overnight. Meanwhile, negative control staining was conducted without primary antibody to exclude false positive fluorescence. After washing with PBS three times, the sections were then incubated with 1:600 Cy3-conjugated goat anti-rabbit (Bioss, Beijing, China) or 1:800 goat anti-mouse IgG DyLight 488-conjugated secondary antibodies (Thermo, IL, USA) for 1 h at 37 °C. Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole; 1:600 diluted in PBS; Solarbio, Beijing, China). The tissue sections were observed under a fluorescent illumination microscope (Olympus IX71, Tokyo, Japan).