Adipose Tissue and Liver Analysis

Sample collection was done as previously described [8 (link)]. Briefly, twenty-four hours after infection, challenged mice were isoflurane anesthetized for retro-orbital blood collection and euthanized by cervical dislocation. Mesenteric adipose tissue (MAT, the visceral adipose tissue between the two peritoneal layers of the mesentery) was removed, its weight was measured, and the tissue was placed in Hanks’s balanced salt solution supplemented with 4% bovine serum albumin (BSA) and 10 mM HEPES buffer (all from Sigma-Aldrich) for further analysis. To minimize variability, all removed mesenteric adipose tissue was used for isolation of adipocyte fraction and stromal vascular fraction cells. Subcutaneous adipose tissue (SAT) and gonadal adipose tissue (GAT) were also removed, their weight was measured, and the latter was preserved in formaldehyde 3.7–4.0% buffered to pH = 7 (Panreac, Darmstadt, Germany) for immunohistochemical analysis. The liver was also collected, some portions were preserved in formaldehyde 3.7–4.0% buffered to pH = 7 (Panreac, Darmstadt, Germany) for immunohistochemical analysis and others were stored in TRI Reagent® (Sigma) for RNA extraction.