Rabbit monoclonal anti occludin antibody

At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan). We used the Image J software to perform the anlysis. Six images of slides were obtained per groups. Images were converted into an 8-bit format, and an intensity threshold was set and kept constant for all images analyzed. Mean uorescence intensities was calculated by dividing the uorescence intensities by the area of outlined regions.

At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4 °C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), and rabbit monoclonal anti-occludin antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).

At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4 °C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), and rabbit monoclonal anti-occludin antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).

At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan).

At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post xation, dehydrated in gradient sucrose solutions of 15% and 30% at 4°C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), rabbit monoclonal anti-occludin antibody (1:100, Abcam), and rabbit monoclonal anti-CD31 antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary uorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a uorescence microscope (Olympus, Japan). We used the Image J software to perform the anlysis. Six images of slides were obtained per groups. Images were converted into an 8-bit format, and an intensity threshold was set and kept constant for all images analyzed. Mean uorescence intensities was calculated by dividing the uorescence intensities by the area of outlined regions.