Plasminogen activator inhibitor type 1 (PAI-1) enhanced 5-chloromethyl-fluoresceindiacetate (CMFDA)-labeled microglial migration
in vivo. The effect of PAI-1 on microglial migration was examined using a stab-injury/cell-injection model. Primary microglia were treated with 1 μg/ml of PAI-1 protein (denatured wild-type protein (control), wild-type protein, R346A mutant protein) for 12 hours. Microglial cells were then labeled with CMFDA and injected into the mouse brain. (A) After 72 hours, three areas (cell-injection site (area 1), an intermediate location between the injection site and the stab-injury site (area 2), and the stab-injury site (area 3) were chosen for the analysis of CMFDA-labeled microglial cell migration (left panel). (A) A schematic diagram of the stab-injury/cell-injection model is also shown (right panel). The stab injury (3 mm long, 2 mm deep) was created 2 mm posterior to the bregma and 4 mm right lateral to the midline. (B) Representative images of CMFDA (green) and Iba-1 (red) staining. Scale bar, 20 μm. (C) Iba-1 microglial staining in the three areas of brain. Scale bar, 200 μm.