Quantitative analysis of gene expression and protein levels

Total RNA was prepared from gonadal fat, liver, or tibialis anterior muscle using TRIzol, then DNase I-treated and reverse-transcribed according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Transcript levels of individual host genes were analyzed by quantitative PCR (qPCR) using the Rotorgene 6000 and assay on-demand TaqMan primer probes and kits, which were normalized to β-actin or RNA polymerase (RNAP), as described (Schertzer et al, 2011 (link); Jorgensen et al, 2012 (link)). Immunoblotting was done in lysates prepared from postclamp tissues (muscle, adipose) and after acute injection of insulin (0.5 IU/kg) into the vena cava (liver), as we described previously (Galic et al, 2011 (link); Hawley et al, 2012 (link)). Formalin-fixed adipose and liver tissues were cut in 8-μm sections by Pathology Laboratory Services at McMaster Children's Hospital. Immunohistochemical (IHC) detection of F4/80 was done using 10 μg/ml rat anti-mouse F4/80 antibody (AbD Serotec, Raleigh, NC, USA) and Vectastain ABC and DAB substrate (Vector Laboratories) and counterstained with Mayer's hematoxylin, as described in Galic et al (2011 (link)).