Storm 24 magnetic bullet blender

To confirm whether systemic dual-LPS administration induced signaling alterations in the mPFC and vHip, protein expression of inflammatory mediator NFκB was examined. This tissue preparation method differed from sample preparation for synaptosome enriched fractions (described below). In this non-behavior tested cohort, the mPFC (n = 4/treatment) and vHip (n = 3/treatment) of C57BL/6J mice treated with LPS (0.83 mg/kg) or vehicle were harvested at 24 h post T = 0 injection as described in Section 2.3 (Supplementary Fig. 1A). Each brain region was homogenized in a Storm 24 magnetic Bullet Blender for 4 min at a speed setting of 4 (Next Advance Inc., Averill Park NY, USA), with 0.5 mm zirconium oxide beads in combination with 50-70 μl of Cell-lytic MT mammalian tissue extraction reagent (Sigma-Aldrich) containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, and 0.1% SDS. The homogenization buffer contained Complete (Roche) protease inhibitor cocktail and phosphatase inhibitor cocktails Type II and III (Sigma-Aldrich). Homogenates were then centrifuged at 16,400 rpm (4°C) for 15 min and supernatants were collected for subsequent SDS PAGE and western blot analysis, as described below (Section 2.5.3).