The polyclonal mouse antibodies raised (-)-Nutlin-3 against putative Inhibitors,Modulators,Libraries inclusion membrane C. pneumo niae GST fusion proteins plus a goat anti mouse IgG conjugated with Cy3 were used to visualize the corresponding antigens. In some cases, the primary antibodies were pre absorbed with either the corresponding or heterologous fusion proteins immobilized onto agarose Inhibitors,Modulators,Libraries beads prior to staining cell samples. The preabsorption approach was carried out by incubating the antibodies with bead immobilized antigens overnight at 4 C fol lowed by pelleting the beads. The remaining supernatants were used for immunostaining. The immunofluorescence images were acquired with an Olympus AX 70 fluores cence microscope equipped with multiple filter sets as described previously.
Briefly, the multi colour labelled samples were exposed under a given filter set at a time and single color images were acquired using a Hamamatsu digital camera. The single color images were then superimposed with the software SimplePCI. All images were processed Inhibitors,Modulators,Libraries using Adobe Photoshop. Background Given that brain depends upon rapid and continuous delivery of oxygen to maintain normal function and sur vival, it may have developed unique molecular responses to hypoxia as compared to other organs and organisms. In spite of the crucial role of oxygen in normal brain function, there have Inhibitors,Modulators,Libraries been relatively few studies of brain hypoxia at the whole genome level. Previous studies using early generation microarrays found changes of expression in whole brain of neonatal rat and in cerebral cor tex of adult mice.
Thus, this study was undertaken in part to examine the brain hypoxia response at the whole genome level in vivo. One of the major goals of this study, however, was to define the regional, whole genome changes of gene expression that could account for the phenomenon of hypoxia preconditioning. In the Inhibitors,Modulators,Libraries first described brain HP model, exposure of neonatal rats to 3 hours of 8% oxygen almost completely protected against hypoxia ischemia induced infarction. In adult mouse brain, hypoxia preconditioning 24 to 48 hours in advance can reduce infarct volume up to 64%. Moreover, the protection provided by HP can last 5 8 weeks. Thus, HP may offer an important platform for the discovery of neuroprotective targets. Though extensively studied, the precise mechanisms accounting for HP remain unknown.
Since HP requires new RNA and protein synthesis, we set out to study the whole genome transcriptional response to HP but in the adult C57BL 6 mouse brain using Affymetrix Expression Arrays. The dynamic gene expression changes were assessed over the following 24 hours after HP a time by which the brain would be protected against focal ischemia. Moreover, the expression changes were assessed on a region by region basis the regional differences of the brain regarding its expression response to hypoxia HP have not been studied systemically.