Alexa fluor 594 goat anti rabbit igg h l

Frozen sections were removed from the freezer, and brain slices were fixed with 4% paraformaldehyde for 15 min, then incubated with a blocking buffer (1X PBS/5% normal goat serum/0.3% Triton X-100, Servicebio Technology Co., LTD, Wuhan, China) for 30 min at room temperature. Sections were subsequently incubated at 4°C overnight with the following primary antibodies: rabbit monoclonal anti-vascular endothelial cadherin (catalogue no. ab33168, dilution, 1: 400; Abcam, Cambridge, MA, USA) and mouse monoclonal anti-Claudin-5 (catalogue no. 35-2500, dilution, 1: 50; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sections were washed 6 times ×5 min with PBS and incubated with the following secondary antibodies: Alexa Fluor 594 goat anti-rabbit IgG (H+L; catalogue no. #8889, dilution,1: 400; Cell Signaling Technology, Danvers, MA, USA), and DyLight 350 conjugated goat anti-mouse IgG (catalogue no. A23010, dilution, 1: 100, Abbkine, Inc., Redlands, CA, USA) for 1 h at room temperature. For Isolectin-B4 staining, slices were incubated with Isolectin-B4 Alexa 488 conjugate (catalogue no. I21411, dilution, 1: 400; Invitrogen; Thermo Fisher Scientific, Inc.) overnight at 4°C. Images were obtained using a fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan). Image processing was performed using Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA, USA).