Further, t test was performed to retain genes with p values less than 0. 05 and a Volcano Plot was generated to identify the two fold differentially expressed genes. The microarray e-book data discussed in this publication is MIAME compliant and has been deposited in NCBIs Gene Expression Omnibus. It is accessible through GEO Series accession number GSE29885. Gene expression profile clustering and pathway analysis Agglomerative average linkage hierarchical clustering of the different experimental groups was obtained for selected groups of genes with GeneSpring GX 7. 3 software with standard correlation used as the similarity matrix. The gene lists obtained was fed into Pathway Studio 6 software to generate pathways for identifying interactions between the genes for validation purposes.
Analysis of gene lists The gene list generated from MATLAB was used to identify Inhibitors,Modulators,Libraries functional groups enriched in the AMC and RMC using DAVID Bioinformatics Database. To identify the Stemness of AMC and RMC, we compared our gene lists to gene lists enriched in embryonic, neural and hematopoietic stem cells. Since the data were accumulated from a different microarray platform, we found orthologs to their genes pertaining to our plat form using the online NetAffyx application. For comparison Inhibitors,Modulators,Libraries of our gene expression data Inhibitors,Modulators,Libraries to that of peripheral blood monocytes, the raw CEL files of monocyte expression data were downloaded from NCBI GEO and the ortho logs pertaining to our platform were identified using the online NetAffyx Inhibitors,Modulators,Libraries application. These files were RMA normalized in Affymetrix Expression Console Version 1.
1 and subsequently the average expression values of the monocyte genes were compared to our microglia gene lists. Double immunofluorescence staining Inhibitors,Modulators,Libraries on postnatal rat brain sections 5 day and 4 week old Wistar rat pups were purchased from the Laboratory Animal Centre, National University of Singapore. The animals were perfused and fixed with 4% paraformaldehyde for further procedure. For double immunofluorescence staining, forebrain sections at 30um were cut through the corpus callosum using cryostat. The sections were incubated with purified mouse anti OX 42 Ig along with rabbit anti ETO or with rabbit anti Dcx or with rabbit anti Sox4 or with rabbit anti Sox11 or with rabbit anti Sept9 with rabbit anti Sept4 overnight at 4 C.
On the following day, the sections were further incubated with either FITC conjugated goat anti mouse IgG or Cy3 conjugated sheep anti rabbit IgG secondary antibody. The sections were counterstained with DAPI and mounted with a fluorescent mounting medium. Photo images were captured using a confocal microscope. Cell culture BV 2 www.selleckchem.com/products/lapatinib.html cells were maintained at 75 cm2 culture flasks in Dulbeccos Modified Eagles Medium supplemented with 10% fetal bovine serum and cultured in 37 C in a humidified atmosphere of 5% CO2 and 95% air incubator. Cells were seeded on 6 well plates at about a density of 1.