Coverslips were mounted on slides and observed using a Zeiss LSM 510 Meta con focal microscope. For AB42s uptake study, the NG2 cell line were incubated with HiLyte Fluor 488 labeled AB42 for the indicated times. Cells were fixed http://www.selleckchem.com/products/SB-203580.html in 4% paraformaldehyde. After permeabilization with 0. 1% Triton X 100, cells were blocked in a solution containing 3% BSA and 0. Inhibitors,Modulators,Libraries 1% Triton X 100 in PBS for 1 hour. Cells were then processed for NG2 immunocytochemistry as described above. For visu alizing lysosomes, the cells were labeled with lysotracker and then fixed, permeabilized and stained with DAPI. Transmission electron microscopy Immunoelectron microscopy was performed as de scribed previously. NG2 cell line treated with AB42 for 6 hours was collected and fixed with 2. 5% glu taraldehyde in 0.

1 M phosphate buffered saline at room temperature for 1 hour and washed 3 times with PBS. The cell pellets were then embedded in 2% agarose II and postfixed with 1% Osmium tetroxide at room temperature for 1 hour. The fixed cell pellets were rinsed three times with distilled water and three times with PBS. The pellets were dehydrated Inhibitors,Modulators,Libraries through an ethanol dilution series up to 100% EtOH and then infiltrated in propylene oxide Epon812 resin mix ture. Then the pellets were infiltrated in 100% Epon812 for 1 hour. Subsequently, the pellets were embedded in 100% Eponate resin and sectioned. Ultrathin sections were treated with 1% sodium periodate for 10 minutes.

Inhibitors,Modulators,Libraries After washed with ddH2O, sections were Inhibitors,Modulators,Libraries blocked in 2% FBS in PBS without Ca2 Mg2 for 30 minutes and incu bated with rabbit anti AB42 IgG antibody overnight at 4 C followed by donkey anti rabbit antibody conjugated to colloidal gold, Jackson Laboratories for 2 hours at room temperature. Sections were double stained with uranyl acetate and lead citrate, and examined under a JEOL JEM1230 electron micro scope. Quantification of AB42 by flow cytometry Quantification of AB42 by flow cytometry was performed as previously described with some modifications. Briefly, primary oligodendrocyte Inhibitors,Modulators,Libraries precursor cells and NG2 cell line were plated at a density of 2 105 cellswell in a 6 well plate overnight. The cells were incubated with HiLyte Fluor 488 AB42 for the indicated times. For nocodazole and cytochalasin D treatment, NG2 cells were preincubated with 150 nM nocodazole and 5 ugml cytochalasin D for 30 minutes and then incubated with HiLyte Fluor 488 AB42 for the indicated times. Cells were removed by the treatment of 0. 01% trypsin, centrifuged at 1,000 g for 5 minutes, and washed with PBS twice. After that, cells were re nevertheless suspended in PBS for the analysis using a FACScan cytometer equipped with a FITC signal detector FL1. Immunoblotting The procedure for immunoblotting was previously de scribed.