Tissue Collection

Healthy C57BL/6J mice were anesthetized in a chamber using isoflurane vapors and sacrificed by cervical dislocation immediately prior to harvesting adipose tissue. Adipose tissue was obtained from six female mice from two age cohorts: three young mice (five months in age) and three aged mice (21 months of age). Mice were sacrificed, tissues were examined and found devoid of gross pathological defects, and adipose tissue was harvested from the omental fat band, uterine fat depot, and the mesenteric fat (Figure 1A). Samples were immediately flash-frozen in liquid nitrogen post-harvest. Before lysis, the tissue was thawed on ice. To remove blood and serum proteins, tissue was washed twice; ice-cold phosphate buffered saline was added to the tissue, gently agitated by vortexing, and centrifuged at 1000 g for five minutes, after which the solution was removed by pipette.

Tissue harvest locations and proteomic workflow. (A) All adipose tissues harvested for this experiment originate in the peritoneal cavity (dotted outline). The omental fat band, mesenteric fat depot, and the left uterine fat depot were harvested from six mice, three old and three young. (B) The 18 tissues were subjected to the proteomic workflow shown. Tissue depots were lysed in SDS and sonicated. Tissues were centrifuged, and the aqueous supernatant was removed and precipitated by methanol-chloroform. Samples were denatured, reduced, and alkylated in gel running buffer. In-gel digestion was performed, resulting in three fractions of peptides per tissue per animal for mass spectrometry analysis