Computational protocol: APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

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Protocol publication

[…] Samples were subjected to quality control analysis by NGSQCtoolkit v 2.3 []. Reads with 70% of their bases having a base quality score ≥ 30 were retained for further analysis. Read alignment and expression estimation was performed using RSEM v 1.2.12 [] with supplied annotations at default parameters against the C57BL/6J mouse genome (build-mm10). Bamtools v 1.0.2 [] were used to calculate the mapping statistics. Differential gene expression analysis between groups was performed using EdgeR v 3.8.5 [,,] following the removal of outlier samples and lowly expressed genes (cpm [or counts per million] < 1 in less than two samples). Normalization was performed using the trimmed mean of M values (TMM). Adjustment for multiple testing was performed using FDR. Genes were considered to be significantly DE at a FDR < 0.05. All raw and processed data is being made available through GEO archives and – Tables. Official ENSEMBL gene IDs for all genes included in this study are provided in column 1 of , and Tables.The Database for Annotation, Visualization and Integrated Discovery (DAVID, []) was used to add functional annotation to DE gene lists and provide statistical assessment of the annotations. We focused on the KEGG pathways []. For each list of DE genes, DAVID determines the number of genes in each KEGG pathway and uses a Fisher exact test to determine the probability that the number of genes in each pathway would have occurred by chance []. A p-value < 0.05 was used to identify significant pathways. Pathways with gene expression changes represented by colors (red–up-regulated, green–down-regulated) were generated at the KEGG website []. [...] For immunostaining involving antibodies for vascular associated proteins, sections were pretreated with Pepsin as previously described [] with minor modifications. Sections were hydrated with H2O for 3 min at 37°C followed by treatment of the tissue with 0.5 mg/ml of Pepsin (Sigma-Aldrich) for 18 min at 37°C. Sections were then rinsed twice with 1X PBS at room temperature (RT) for 10 min. After Pepsin pretreatment, sections were rinsed once in 1X PBT (PBS + 1% Triton 100X) and incubated in primary antibodies diluted with 1X PBT + 10% normal goat or normal donkey serum over two nights at 4°C. After incubation with primary antibodies, sections were rinsed three times with 1X PBT for 10 min and incubated for two hours in the corresponding secondary antibodies (1:800, Invitrogen). Tissue was then washed three times with 1X PBT for 10–15 min, incubated with DAPI and mounted in Poly aquamount (Polysciences). The following primary antibodies were used: goat anti-COL4 (1:40, R&D), goat anti-PDGFRβ (1:40, R&D), goat anti-CD31 (1:40, R&D), rabbit anti-LAM (1:200, Sigma-Aldrich), goat anti-mouse APOE (1:50, Santa Cruz Biotech), Biotinylated Lycopersicon Esculentum (Tomato) Lectin (1:200, Vector), rabbit anti-IBA1 (1:200, Wako), rabbit anti-GFAP (1:200, Dako), rabbit anti-AQP4 (1:200, Sigma-Aldrich), mouse anti-synaptophysin (SYN, 1:200, Millipore), mouse anti-NeuN (1:300, Millipore), rabbit anti-fibrinogen (FIBRIN, 1:200, DAKO). Blocking serum was not included in primary antibody solutions that contained the fibrinogen antibody.For quantitative analysis of FIBRIN and SYN in the cortex, four images were randomly taken in the parietal cortex for each brain for each mouse and opened in ImageJ (1.47 d) software as a black and white image as reported previously []. Stained intensity and % of area statistics were obtained by generating surface segmentation using the same threshold criteria for all the pictures. For quantification of PDGFRβ+ or IBA1+ cells in the cortex and CA1, four images were taken for each brain from each mouse with a Zeiss Axio Imager fluorescent microscope and manually counted using the cell counter plugin from the ImageJ (1.47 d) software. For quantification of pericyte coverage of microvessels, the length PDGFRβ+ pericytes were calculated using the NeuronJ plugin in ImageJ (1.47d) software. All image analyses were performed blind to the experimental conditions. […]

Pipeline specifications

Software tools ImageJ, NeuronJ
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases Alzheimer Disease, Thoracic Outlet Syndrome, Neurodegenerative Diseases