Journal: Nature neuroscience
Article Title: Epigenetic regulation of brain region-specific microglia clearance activity
Figure Lengend Snippet: cbMg display a cell clearance phenotype (a) CD68+ lysosome content (red) in IBA1+ microglia (green) in brain sections from 4/6mo wild-type mice (DAPI: blue). Scale: 10 μm. Representative image (left); quantification of lysosomal area/microglia area (right) are shown. stMg: mean=0.02437, SEM=0.01011; cbMg: mean=0.1424, SEM=0.02527; p=0.0166, F=6.245, t 2 =7.668; > 15 images/region from n=3 mice. (b) DAPI content (blue) in CD68+ lysosomes (red) of IBA1+ microglia (green) was determined using 3D reconstruction (Imaris) from 4/6mo wild-type mice. Scale: 5 μm. Representative reconstruction with side scatter views (left); quantification of percentage of microglia with DAPI+ CD68+ lysosomes (right). Arrows indicate DAPI+/CD68+ lysosome, which is shown with a zoomed in view. 3D axes are shown. stMg: mean=0.1675, SEM=0.06029; cbMg: mean=0.6960, SEM=0.0868; p=0.0300, F=2.077, t 2 =5.639; > 200 cells/region from n=3 mice. Bar graphs with individual data points show mean ± SEM, t-tests were two-tailed paired. (c) Schematic showing microglia-specific TRAP-sequencing. (d) Heatmap with hierarchical clustering distances shows the variation in the expression levels (z-scored log2 RPKM [z-score]) of 733 stMg- and 297 cbMg-enriched genes identified by TRAP (DESeq2, n=2/age/region). Selected Gene Ontology (GO) annotations (Enrichr) enriched for stMg-/cbMg-enriched genes are shown. y-axis: -log10 (p-value). Dotted lines: p=0.05. (e) Genome browser views (IGV) of normalized read counts of selected genes in stMg/cbMg by TRAP.
Article Snippet: Primary antibodies: GFP (1:2000, ab6556 and 1:500 ab13970 Abcam, Cambridge, MA) , , IBA1 (1:500, 019-19741, Wako Chemicals, Richmond, VA) , P2RY12 (1:5000, kind gift from O. Butovsky) , CD68 (1:250, MCA1957, Biorad, Hercules, CA) , NeuN (1:500, MAB377, EMD Millipore, Billerica, MA) , GFAP (1:500, G3893, Sigma) , OLIG2 (1:250, MABN50, EMD Millipore) , CD11b (1:1000, MCA711GT, Biorad, Hercules, CA) , H3K27me3 (1:500, C36B11, Cell Signaling) , CD74 (1:50, sc-5438, Santa Cruz) , MHCII (1:200, ab23990, Abcam) , ApoE (1:100, AB947, Millipore) , AXL (1:100, AF854, R & D Systems) , cCASP3 (1:400, #9661, Cell Signaling) and Ki67 (1:200, ab16667, Abcam) .
Techniques: Mouse Assay, Two Tailed Test, Sequencing, Expressing, Significance Assay