Brain slicer matrix

Microarray analysis was performed on hypothalamic areas of mice fed a ND or a HFD for 4 or 8 weeks as described^{66 (link)}. Briefly, hypothalamic area was dissected using the brain slicer matrix (Zivic Instruments), and total RNA was isolated with RNeasy Mini kit/RNeasy Lipid tissue mini kit (Qiagen). Probes were prepared using NuGEN Ovation Pico WTA V2 kit and NuGEN Encore Biotin Module, and hybridized to Rat and Mouse Gene 1.0 ST GeneChip arrays (Affymetrix). Arrays were scanned using an Affymetrix GCS3000 scanner and Affymetrix Command Console software, and data were normalized using the RMA algorithm in Affymetrix Expression Console. Microarrays were normalized for array-specific effects using Affymetrix’s “Robust Multi-Array” (RMA) normalization and were reported on a log₂ scale. For statistical analyses, we removed all array probe sets in which no experimental groups had an average of log₂ intensity >3.0. Linear models were fit for each gene using the Bioconductor “limma” package in R^{67 (link)}. Moderated t-statistics, fold change, and the associated P-values were calculated for each gene. To account for the fact that thousands of genes were tested, we reported FDR-adjusted values, calculated using the Benjamini–Hochberg method⁶⁸ . Pathway analysis was performed using the GO enrichment and Ingenuity IPA analysis.

Animals were humanely killed 72 h after exposure to HI or sham treatment. Blood was sampled via cardiac puncture, which was allowed to coagulate and was centrifuged (1200 g, 5 min) to acquire the serum. Serum samples were stored at −80 °C until analysis. Brains were perfused with PBS, paraformaldehyde (PFA, 4%) and then weighed. Brains were placed in 4% PFA for 24 h, then stored in phosphate-buffered sucrose solution (30%). Brains were sectioned into 2 mm coronal segments using a brain slicer matrix (Zivic instruments, Pittsburgh, PA, USA), then each segment was placed in a plastic mold, covered in optimal cutting temperature compound (OCT) and frozen by placing the mold into isopentane on a bed of dry ice. Frozen brains were stored at −80 °C, until sectioned on a cryostat (CM3050S, Leica, Nusslock, Germany) at 20 μm and mounted on gelatin-coated slides.