After sacrifice, animals were perfused with ice cold 0. 9% PBS, the brain removed and dissected in two by mid sagittal dissection. One was immediately stored at ?80 C for biochemistry assays or total RNA extraction, and the other immediately immersed in 4% paraformaldehyde in 0. 1 M PBS overnight for immunohistochemis selleckbio try studies. Immunohistochemistry Immunostaining was performed as reported previously by our group. Serial 6 um thick coronal sections were mounted on 3 aminopropyltriethoxysilane coated slides. Every eighth section from the habenular to the posterior commissure was examined using unbiased stereological principles. The sections for AB were deparaffinized, hydrated, pretreated with formic acid and subsequently with 3% hydrogen peroxide in methanol.

The sections for glial acidic fibrillary protein and CD45 were deparaf finized, hydrated and treated with 3% hydrogen peroxide in methanol and subsequently antigen retrieved with cit rate. Sections were blocked in 2% fetal bovine serum before incubation Inhibitors,Modulators,Libraries with primary antibodies overnight at 4 C. Subsequently, sections were incubated with biotinylated anti mouse IgG and then developed using the avidin biotin complex method with 3,3 diami nobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by AB immunoreactivity using the software Image Pro Plus for Windows version 5. 0. The threshold optical density that discrimi nated staining from background was determined and kept constant for all Inhibitors,Modulators,Libraries quantifications.

The area occupied by AB immunoreactivity was measured by the software and divided Inhibitors,Modulators,Libraries by the total area of interest to obtain the percentage area of AB immunoreactivity. Biochemical analyses Mouse brain homogenates were sequentially extracted first in radioimmunoprecipitation assay for the AB soluble fractions and then in FA for the AB insoluble fractions as previously described. AB1 40 and AB1 42 levels were assayed by a sensitive sandwich ELISA kits. Inter leukin 1 B levels in brain homogenates were assayed Inhibitors,Modulators,Libraries by a specific and sensitive sandwich ELISA kit, following the manufacturers instructions. Supernatants from the cell cul ture experiments were also assayed Inhibitors,Modulators,Libraries for their levels of lactate dehydrogenase by a colorimetric assay kit. inhibitor Nutlin-3a Analyses were always performed in duplicate and in a coded fashion. Western blot analyses RIPA extracts from brain homogenates were used for western blot analyses.