Intracortical cell injection was performed using a 26 G needle through a guide cannula with a flow rate of 0. 1 ul min 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. Iba 1 immuno fluorescence staining was performed selleck chem inhibitor as described above for immunohistochemistry, except the secondary antibody Inhibitors,Modulators,Libraries was donkey Cy3 conjugated anti rabbit antibody. The sections were mounted on gelatin coated slides and allowed to air dry overnight. Data acquisition and immunohistological intensity measurement The level of coronal sections that passed the striatum was determined in accordance with a mouse brain atlas.
Tiled images of each section were captured with a charge coupled device color video camera through a 100 �� objective lens attached to a microscope. A composite of the images was Inhibitors,Modulators,Libraries then constructed for each section with Photoshop CS3. Immunohistological intensity analysis of Iba 1 staining was performed as previously described. Composite images of stained sections were fast Fourier transform band pass filtered to eliminate low frequency drifts and high frequency noises. The image was set with a binary threshold of 50% of the background level, and then the particles were converted to a subthreshold image area with a size of 20 to 300 pixels, which was judged as showing the Iba 1 positive cells. This range was obtained from the analyzed size of Iba 1 positive cells from six sec tions for each animal.
To count Inhibitors,Modulators,Libraries the Iba 1 positive cells, five squares were placed around the injec tion site in the subthreshold image of the six independent sections, and the cells in the five squares were counted and statistically analyzed. Phagocytosis of fluorescent zymosan particles BV 2 microglial cells were seeded at a density of 7. 5 �� 104 cells well in 24 well plates. Cells were treated with the recombinant human PAI 1 protein, mouse PAI 1 protein, BSA, monoclonal anti mouse TLR2 antibody, polyclonal anti mouse integrin B3 antibody, and vitronectin for 1 hour in serum free Inhibitors,Modulators,Libraries DMEM. Cells were then incu bated at 37 C for 3 hours with 30 ug ml of fluorescent zymosan particles BioParticles conjugated with Alexa Fluor 594, Mo lecular Probes Inc.
Primary microglia cultures were similarly treated with mouse Inhibitors,Modulators,Libraries PAI 1 or RAP for 1 hour, and then incubated with 30 ug ml of zymosan particles for 90 minutes. Cells were then washed five times with ice cold PBS to remove bound particles. Photomicrographs of five randomly chosen fields were taken in three separate experiments. A minimum of 400 microglial cells per well were counted, and the percentage of phagocytic cells was determined as previously described. Recent reports have selleck inhibitor indicated that washing three to five times with ice cold PBS effectively removed extracellular bacteria and zymosan particles.