All images were acquired at the same exposure and were automati c

All images were acquired at the same exposure and were automati cally aligned using the stitching tool in the Axiovision LE software. Once acquired, kinase inhibitor Y-27632 all images were opened in ImageJ and were normalized the threshold was set for each image using the histogram mean at the same stan dard deviation. Each image was adjusted to the thresh old and set to scale in pixels. The parameters measured include the area, integrated density, perimeter, and fer ets diameter for each plaque. To help eliminate back ground the particle size pixel was set at 30 infinity pixel. To quantify plaques, the brain level of cut sections was fixed for all mice at the region of the motor cortex and hippocampus corresponding to the starting section at interaural 2. 34 mm and Bregma 1. 46 mm of The Mouse Brain Atlas by George Paxinos and Keith Franklin.

A fixed thickness of 16 uM coronal sections Inhibitors,Modulators,Libraries at regular Inhibitors,Modulators,Libraries intervals was maintained in all animals. The amyloid pla ques were quantified from throughout the sections from five sections per mouse and mean values were generated for each mouse. Pictures were montaged and, for quanti fication by image J software, the color images were Inhibitors,Modulators,Libraries con verted in to HSV format and 8 bit channels. Plaques Inhibitors,Modulators,Libraries were quantified in an unbiased manner by an investigator blind to the treatment nature of the samples. Plaque bur den was calculated as the area occupied by the plaques divided by the total brain region area. The data are expressed as percent change in means from the controls. Quantitation of Ab40 levels in the brain by ELISA Ab40 levels in the brain extracts were determined by sand wich ELISA.

Briefly, the wet mass of the brain was weighed and homogenized thoroughly in cold 1% CHAPSOPBS with protease inhibitors. The homogenate was ultra centri fuged at 100,000 g for 60 minutes. The samples were further diluted to 40 fold and stored on ice until use. The Ab standard was dissolved in hexafluoroisopro panol at 1 mgml, sonicated and dried under Inhibitors,Modulators,Libraries nitrogen. The dried Ab40 was resuspended in DMSO, separated into ali quots and frozen at 80 C. The rest of the protocol is exactly as described previously from our laboratory. The quantity of Ab40 in each sample was measured in quadruplicate. Total protein concentrations were deter mined using the BCA assay. Iba1 I mmunohistochemistry Brain sections from saline and BCNU treated mice were washed two times with PBS 1X for five minutes.

Antigen retrieval was carried out by immersing slides in 10 mM citric acid for 10 minutes at 90 C. Sections were washed three times with PBS 1X for five minutes and incubated in blocking solution for one hour at RT. The sections were incubated overnight with anti Iba1ALF1 mouse monoclonal NSC639966 antibody in blocking solution at 4 C. After washing three times in PBS 1X for 5 minutes, the sections were incubated with Alexa Fluor 568 goat anti mouse IgG in blocking solution at RT for two hours in the dark.

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