Goat anti mouse igg dylight 488 conjugated secondary antibodies

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).

Immunofluorescent staining of HIF‐1α, SP1 and Robo4 was performed using OCT‐embedded sections or cultured ARPE‐19 cells on polylysine‐coated glass coverslips after culture under hyperglycaemic conditions. Slides were rinsed, and cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100 in PBS for 15 minutes at room temperature, and blocked (1% bovine serum albumin in PBS) for 30 minutes at 37°C. Subsequently, sections were treated with primary antibodies at 4°C overnight, including rabbit polyclonal anti‐HIF‐1α (1:50 dilution; Affinity), rabbit polyclonal anti‐SP1 (1:100 dilution; Affinity), and rat monoclonal anti‐Robo4 antibodies (1:50 dilution; Santa Cruz Biotechnology). Slides were then incubated with Cy3‐conjugated goat anti‐rabbit (Bioss, Beijing, China) or goat anti‐mouse IgG DyLight 488‐conjugated secondary antibodies (Thermo, IL, USA) for 1 hour at 37°C. Nuclei were stained with DAPI (1:600 diluted in PBS; Solarbio, Beijing, China). The slides were observed under a fluorescent illumination microscope (Olympus IX71, Tokyo, Japan).