Ab65842

Glucose (Glc) and triglycerides (TG) were assessed in blood-derived serum using Liquick Cor kits (Cormay, Lomianki, Poland) according to the manufacturer’s instructions. Insulin concentrations were measured by a mouse insulin ELISA kit (Invitrogen, Thermofisher, Waltham, MA, USA) according to the company’s instructions. The insulin sensitivity was assessed by calculating the quantitative insulin sensitivity check index (QUICKI), as previously reported [32 (link)]. For the determination of the inflammatory parameter C-reactive protein (CRP), and protein carbonylation and nitration, known markers of oxidative stress, slot-blot was performed. In brief, equal volumes of serum samples from animals of each group were slot-blotted under vacuum in a nitrocellulose membrane (Amersham™ Protan™, GE Healthcare Lifesciences) and incubated for 1 h with a primary antibody (rabbit anti-CRP, 1:1000, ab65842, Abcam, Cambridge, UK; mouse anti-nitrotyrosine (nitroTyr), MAB5404, Millipore Sigma, St. Louis, MO, USA; mouse anti-dinitrophenylhidrazone (DNP), 1:1000, MAB2223, Millipore Sigma). Incubation with secondary antibody and signal detection are detailed in Section 2.9.

For detecting serum amyloid P component (SAP) or C-reacting protein (CRP) in mouse plasma, 0.4 or 2 μL of plasma diluted with 14.6 or 16 μL of PBS and 5 or 6 μL of 4x Laemmli Sample Buffer (Bio-Rad Laboratories, CA, USA) was sonicated for 15 min and boiled for 10 min at 95 °C. This mixture was subjected to Western blotting (see “Immunopreciptation and Western blotting”) using antibodies against SAP (1:1,000, AF2558, Bio-Techne Corporation/R&D Systems, MN, USA) and CRP (1:200, ab65842, Abcam, MA, USA). Concentration of CRP in human plasma was measured by Human C Reactive Protein ELISA Kit (Abcam, MA, USA) according to manufacturer’s protocol. Samples were diluted 1:50,000 and measured in duplicates.