Intras selleck kinase inhibitor triatal injection of the vehicle or recombinant human PAI 1 protein of wild type or R346A mutant was performed using a 26 G nee dle. Denatured PAI I protein, which was used as a control, was prepared by heating for 15 minutes at 95 C. The flow rate of the injection was 0. 1 ulmin maintained by a microsyringe pump. After re moving the needle, the skin was sutured with 6. 0 mm silk thread. The mice were killed 48 hours after the injection. Immunohistochemistry Mice were anesthetized with ether, and transcardially perfused with 4% paraformaldehyde in PBS. Brains were post fixed and cryoprotected with 30% sucrose solution for 24 hours. The fixed brains were embedded in opti mal cutting temperature compound and then cut into 12 um thick coronal sections on a cryostat. The tissues were permea bilized in 0.

1% Triton X 100, and blocked with 1% BSA and 5% normal serum. After washing with PBS, the sec tions were incubated at 4 C overnight with rabbit poly clonal Iba 1 antibody. The sections were then incubated with biotiny lated anti rabbit IgG antibody. Subsequently, the sections were incubated with avidinbiotin Inhibitors,Modulators,Libraries com plex reagents for 30 minutes at room temperature, followed by detection with diaminobenzidine. Stab injury and cell injection assay To evaluate in vivo microglial cell migration, we used a stab Inhibitors,Modulators,Libraries wound injury model as described previously. ICR mice were anesthetized by intra peritoneal injection of tiletaminezolazepam 30 mgkg and xylazine 10 mgkg, and positioned in a stereo taxic apparatus, on a homeothermic heat blanket at 37 C to maintain normal Inhibitors,Modulators,Libraries body temperature during surgery.

The skull was exposed by a sagittal skin inci sion, and a small hole was drilled through the skull. The guide cannula was implanted at 4 mm lateral from the bregma, and 3 mm below the skull using a 22 G needle, and cemented. Inhibitors,Modulators,Libraries After 3 days, the skull bone located at 2 mm posterior from Inhibitors,Modulators,Libraries the guide can nula was thinned with a high speed drill, and sellectchem then a 32. 50. 1 mm sterilized razor blade was stereotaxic ally inserted to a depth of 3 mm below the skull to create a coronal stab injury, and immediately removed. After re moving the blade, the bone was covered. Primary microglial cells were incubated with 1 ugml of recombin ant human PAI 1 proteins in six well plates for 12 hours, and labeled with 5 umoll CMFDA for 15 minutes. Intracortical cell injection was performed using a 26 G needle through a guide cannula with a flow rate of 0. 1 ulmin using a microsyringe pump. After sur gery, skin was sutured with 6. 0 mm silk thread. At 72 hours after the injection, the mice were killed. Migration of CMFDA labeled microglial cells was esti mated using immunofluorescence assay.