Laser capture microdissection Whole fresh brains were removed from 5 day postnatal Wistar rat pups and 4 week old Wistar rats and placed in liquid nitrogen immediately for a short time and then frozen in a cryostat. The forebrain was sectioned coronally through the CC at 5 um thickness and tech support mounted on precleaned slides. The sections were fixed in 75% ethanol for 1 min and incubated with peroxidase conjugated isolectin for 15 min. The sections Inhibitors,Modulators,Libraries were then dehydrated by a graded series of ethanol and cleaned in xylene. The slide was placed on the microscope stage of MMI CellCut. The 4 X, 10 X to 40 X objective lenses were used to achieve the proper placement of the cap above the CC. Lectin stained microglia cells were selected and cut by laser and collected into the cap of tube.

Extra care Inhibitors,Modulators,Libraries was taken to minimize the contamination of materials from other cell types while laser dissecting microglia from the CC. Microarray analysis Total RNA was extracted from 600 isolated microglia cells per group using RNeasy micro kit, quantified by Nanodrop 1000 Inhibitors,Modulators,Libraries and hybridized to each microarray chip. RNA was reverse transcripted into the first strand cDNA using a T7 Oligo Primer. After second strand cDNA synthesis, the double stranded cDNA was purified and served as a template in the first cycle of in vitro tran scription reaction. The unlabeled cRNA was then reverse transcripted into the first strand cDNA of the second cycle using random primers. Subsequently, the T7 Oligo Promoter Primer was used in the second strand cDNA synthesis to generate double stranded cDNA template containing T7 promoter sequences.

Then the double stranded cDNA was amplified and labeled using a biotinylated nucleotide analog ribonu cleotide mix in the second IVT reaction. The labeled cRNA was then cleaned up, fragmented, and hybridized Inhibitors,Modulators,Libraries to Rat Genome 230 2. 0 Array. A total of six arrays were carried out in the present study. The arrays were stained according to the manufacturers protocols and then scanned with the Genechip scanner. Initial analysis of the scanned images Inhibitors,Modulators,Libraries was performed by GeneChip Operating Software. For absolute analysis, each chip was normalized to a target intensity of 500, and probe sets were assigned a signal intensity and de tection call of Present, Marginal or Absent. Data analysis and generation of gene lists The absolute data were exported into GeneSpring GX 7.

3 software. All the six chips were globally normalized and the genes of over 2 fold differential expression were filtered out and used for functional analysis. Data normalization and generation of gene lists using MATLAB Raw CEL files of the six chips were neither RMA normalized using the Affymetrix Expres sion Console Version 1. 1. The normalized data was then used to identify differentially expressed genes between AMC and RMC in MATLAB R2009a. For the statistical ana lysis, we used the Exploring Gene Expression Data demo scripts in the Bioinformatics Toolbox.