bio rad hercules calif samples were denatured in a sample buffer invitrogen carlsbad calif for 10 min at 70 u00b0 c fifteen u03bcg of protein was loaded per well and proteins separated under non reducing conditions on nupage 4 12 novex bis tris gels invitrogen for 60 min at 200 v and then blotted onto pvdf membranes bio rad for 120 min at 30 v for the detection of app and gfap protein membranes were blocked in phosphate buffered saline ph 7 4 and 0 05 tween 20 pbs t with 5 non fat dried milk and thereafter incubated with mouse monoclonal anti alzheimer precursor protein 22c11 a4 antibody 1 1000 chemicon temecula calif and rabbit anti gfap antibody 1 1000 promega madison wis in pbs t containing 1 non fat dried milk over night at 4 u00b0 c to standardize the amount of protein loaded on to each gel the polyclonal rabbit anti b actin antibody 1 1000 cell signaling technology danvers mass was used the membranes were washed three times for 15 min in pbs t and then incubated with secondary horseradish peroxidase conjugated antibodies anti mouse igg and anti rabbit igg jackson immunoresearch laboratories inc west grove pa at rt for 1 hr three final washes of 15 min in duration each were performed signals were visualized by incubation of the membranes in ecl plus reagents amersham biosciences buckinghamshire uk exposed to hyper performance chemiluminescence film amersham and developed according to standard procedures the films were scanned and the optical density of each specific band relative to u03b2 actin was analyzed by the public domain nih image j software version 1 33u and expressed in means u00b1 sd from experiments performed in triplicate total rna from hippocampal and cortical tissues from each animal treated with either phenserine.