Proteins were extracted from CSF samples using a 2D cleanup kit (GE, 80-6484-51), resuspended in 100 μl of lysis buffer (30M Tris pH 8.5, 7M Urea, 2M Thiourea, 4% CHAPS) and quantitated by Bradford assay using BSA as a standard. Cy-dye labeling, isoelectric focusing and gel electrophoresis of 2D-cleaned CSF samples were performed according to standard DIGE protocols.31 (link) Labeled samples were combined (1 Cy3, 1 Cy5 and 25 μg of Cy2 sample/gel) and diluted 2X with rehydration buffer [7M Urea, 2M thiourea, 2% CHAPS, 1% pH 3-10 IPG buffer (GE Healthcare), 50mM DTT, 1% saturated bromophenol blue solution] to a final volume of 450 μL and then isoelectric focused on an IPGphor II (GE Healthcare) instrument using standard isoelectric focusing protocols for pH 3-10 strips (GE Healthcare). Finally, pH strips were reduced and alkylated, placed in 20x24cm 12% SDS-PAGE gels, and run in a Dalt-12 electrophoresis system (GE Healthcare) at 2 watts per gel for 45 minutes, followed by 15 watts per gel until the dye front reached the bottom (~4 hours).