Intrastriatal injection of human plasminogen activator inhibitor type 1 protein Mice were anesthetized by intraperi toneal injection of tiletamine zolazepam and xyla zine and positioned in a stereotaxic apparatus. The mice were placed on a homeothermic heat blanket at 37 C to maintain normal body temperature dur ing surgery. The skull was exposed by a skin incision, and a small hole was MLM341 drilled through the skull. To avoid pas sing through the ventricles, the guide cannula was implanted at the stereotaxic coordinates of 1 mm anterior to the bregma, 2 mm lateral to the bregma, and 4 mm below the skull using a 22 G needle, and cemented. Intras 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 Inhibitors,Modulators,Libraries PAI I protein, which was used as a control, was prepared by heating for 15 Inhibitors,Modulators,Libraries minutes at 95 C. The flow rate of the injection was 0. 1 ul min 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 Inhibitors,Modulators,Libraries 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 avidin biotin 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 tiletamine zolazepam 30 mg kg and xylazine 10 mg kg, and positioned in a stereo taxic apparatus, on a homeothermic heat blanket at 37 C to maintain normal 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 Inhibitors,Modulators,Libraries the skull using a selleck chemical Ceritinib 22 G needle, and cemented. After 3 days, the skull bone located at 2 mm posterior from the guide can nula was thinned with a high speed drill, and then a 3 �� 2. 5 �� 0. 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.