Lithium heparin microtubes

As described above, eight nonsibling animals per group were sampled at each of the three time points. Animals were housed in metabolic caging for 24 h with free access to food and water, during which time urine was collected and food and water intake measured. After removal from metabolic cages, animals were killed by rising concentration of CO₂ followed by cervical dislocation. Blood was collected by cardiac puncture into lithium-heparin microtubes (Sarstedt, Leicester, UK), centrifuged at 830 g for 10 min, and stored at −20°C prior to analysis. All major organs were dissected from the carcass, with kidneys separated into cortex and medulla. The wet weight of organs and gonadal and perirenal fat depots were recorded. Tissues were snap frozen in liquid nitrogen and stored at −80°C prior to analysis or fixed in 4% formalin.

After 20 weeks of treatment, the mice were euthanized by isoflurane (Primal Critical Care, Bethlehem, PA, USA) overdose and their brain tissues were harvested following blood collection for immunofluorescent staining and immunoblotting analysis. For the purposes of immunostaining, the mouse brains were collected after perfusion with ~15 mL of ice-cold 1X PBS followed by ~15 mL of an ice-cold 4% paraformaldehyde (PFA) (#15714-S, Electron Microscopy Sciences, Hatfield, PA, USA) solution. The brains were postfixed in 4% PFA for 24 h at 4 °C. The brains were transferred to 30% sucrose for 5 days at 4 °C. After dehydration, the brains were embedded in a Tissue-Tek optimal cutting temperature compound (OCT), flash frozen, and stored at −80 °C until used for cryminosectioning. Hippocampal tissues of WT (male N = 6; female N = 6) and PS19 (vehicle-treated male N = 6, vehicle-treated female N = 6; D-DPTIP-treated male N = 6, D-DPTIP female N = 6) mice were harvested fresh and stored at −80 °C until use. The blood was collected via cardiac puncture and placed into lithium heparin microtubes (#41.1393.105, Sarstedt, Nümbrecht, Germany). Plasma samples were collected from the blood by centrifugation at 500× g for 10 min and stored at −80 °C until bioanalysis.

Whole blood for determination of serum analytes (triglycerides and insulin) was collected into microtubes with coagulation activator (Sarstedt, Nümbrecht, Germany) and left to clot before being centrifuged (15 000 g for 2 min), and the serum was frozen at −80°C. Whole blood was added to lithium heparin microtubes (Sarstedt, Nümbrecht, Germany) containing ethylene glycol tetraacetic acid–glutathione and tetrahydrolipostatin, gently mixed, and the plasma was removed and frozen at −80°C until analysis of NEFA. Triglycerides were analysed using enzymic photometry (ABX Pentra 400, HORIBA Medical, Montpellier, France) and insulin concentration using a solid‐phase ¹²⁵I human‐specific radioimmunoassay (Merck Millipore, Billerica, MA, USA). NEFA was analysed by the ACS‐ACOD Method (Wako Diagnostics, Richmond, VA, USA).