Horseradish peroxidase hrp conjugated goat anti rabbit igg secondary antibodies

Paraffinized brain sections were dewaxed and dehydrated in xylene and ethanol solution, followed by antigen retrieval and blocking with 10% goat serum (Beyotime Biotechnology Co., Ltd.) at room temperature for 15 min. All sections were then incubated with anti-VEGFR2 antibody (1:800 dilution; cat. no. 9698S; Cell Signaling Technology, Inc.) at 4˚C overnight. Subsequently, the sections were washed in PBS three times and incubated with Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:200 dilution; GB23303; Wuhan Servicebio Technology Co., Ltd.) for 10 min at room temperature. Next, the sections were visualized using a diaminobenzidine kit (Wuhan Servicebio Technology Co., Ltd.) and counterstained with hematoxylin. With a light microscope, an investigator blinded to the experimental groups randomly chose three separate tissue sections for each rat (four rats for each group). The staining was analyzed using ImageJ analysis software1.50i (NIH).