Cy3 labeled secondary antibody goat anti rabbit igg

For immunofluorescent staining, brain slices were immersed in the EDTA-antigen retrieval solution (PH 8.0, BioGenex, Fremont, California, USA) for 4 min with intermittent microwave irradiation (4 s on/3 s off at 200 W). Permeabilize and block the tissue with 0.5%Triton X-100 in PBS with 3% BSA at room temperature for 1 h. Rinse specimens with PBS several times; incubation then was performed with one of the primary antibodies: rabbit anti-CD31 (1:50, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 16 h. Rinse specimens with PBS several times; brain slices were incubated with Cy3-labeled secondary antibody (goat anti-rabbit IgG, 1:100, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h, away from light. Images were acquired by confocal microscopy (Olympus BX53, Tokyo, Japan). Three visual fields at 100 × magnification around the hematoma from three brain slices of each mouse were selected for statistical analysis. Microvessel density was evaluated by a number of microvessels in each field. Image analysis was performed with the Image J software. In the slices immunostained for CD-31, immunepositive cells were counted by an investigator who was blinded to different treatment groups in six fields (cells/mm 2 ) in the median part at 0 to 0.5 mm around the hematoma.