J Cachexia Sarcopenia Muscle (2011) 2:181–188 the course of the study. As has previously been described [4], donor mice were used to establish and maintain a tumour line, which consistently produced tumour growth (MAC13) (n = 3) and tumour growth with cachexia (MAC16) (n = 4). Tumour fragments grown and maintained in donor mice were dissected and re-implanted subcutaneously into the flank of recipient mice with frequent monitoring and recording of body weight and tumour size. With cachectic weight loss evident at approximately 9–12 days post implantation, mice were anaesthetised, using pentobarbital sodium (70 mg/kg), for tissue collection before weight loss exceeded 25% of original body weight or before tumour growth exceeded 1,000 mm3. Skeletal muscle tissue was collected, weighed and immediately snap-frozen in liquid nitrogen and stored at −80°C for later use. 2.2 Detection of O2− by DHE fluorescence staining Skeletal muscle O2− was measured using O2−-sensitive Dihydroethidium (DHE) dye. Cell-permeable DHE reacts with O2−, converting DHE into ethidium fluorescence. DHE (5 μM) was applied to quadriceps cross-sections (5 μm) and incubated in a light-protected oven at 37°C for 30°min. The sections were washed with PBS to remove excess DHE, and fluorescence was assessed by way of fluorescence microscopy (Axiocam HBO 50/AC, Zeiss, Germany). The muscle was analysed in three sections to obtain measurements from the whole tissue. Ethidium fluorescence density was detected from the whole section with MCID imaging software (Imaging Research Inc, Australia) and expressed as arbitrary units of fluorescence. 2.3 Reverse transcription real-time PCR RNA was extracted from frozen quadriceps muscle using Tri Reagent (Molecular Research Centre), according to the manufacturer's protocol. Total RNA concentration was determined spectrophotometrically at 260 nm. Prior to reverse transcription (RT), all RNA samples were DNase- treated (Promega), and first-strand cDNA was generated from 1-μg RNA using AMV RT (Promega). Pre-designed TaqMan Gene Expression Assays (Applied Biosystems) were used containing specific primers and probes for the genes of interest. Real-time PCR was performed using Applied Biosystems 7500 detection system and PCRs were performed using TaqMan Gene Expression Master Mix (Applied Biosystems). To compensate for variations in input RNA amounts and efficiency of reverse transcription, GAPDH mRNA was quantified and all results were normalised to these values. Fluorescent emission data were captured and mRNA levels were analysed using the CT value [17]. 183 2.4 Protein electrophoresis and western blotting Protein was extracted from frozen quadriceps muscle homogenised in ice-cold radio-immunoprecipitation assay buffer containing Tris–HCl (50 mmol/L; pH 7.4), NaCl (150 mmol/L), NP-40 (1%), sodium deoxycholate (0.5%) and SDS (0.1%) and centrifuging at 13,000×g for 15 min at 4°C, to remove insoluble material. The protein concentra- tion was determined by the Bradford method (Bio-Rad) and equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 2 h at room temperature in Tris-buffered saline containing Tris–HCl (20 mM; pH 7.6) NaCl (137 mM) and Tween 20 (0.1%) with 5% BSA and probed with primary antibodies for either NOX2 (gp91phox), p40phox, p67phox or GAPDH (1:200; Santa Cruz Biotechnology) overnight at 4°C. Antibody binding was detected using horseradish peroxidase-conjugated secondary antibody (1:50,000; Santa Cruz Biotechnology). The protein bands were detected by SuperSignal West Dura chemilumi- nescence reagents (Thermo Scientific). The LAS 4000 Imaging System (Fujifilm Life Science, USA) was used to visualize protein bands, and densitometry was performed with MultiGauge software (Fujifilm Life Science, USA). To compensate for variation in protein loading, the relative density of immunoreactive bands were normalised to the density of the corresponding bands for GAPDH. 2.5 Antioxidant enzyme activity and hydrogen peroxide assays Spectrophotometric assay kits were used to measure SOD (Cayman-706002), catalase (Cayman-707002) and GPx (Cayman- 703102) activity and hydrogen peroxide (Cayman- 600050) levels, in muscle homogenates. Frozen muscle pieces (100 mg) were placed in ice-cold HEPES buffer (20 mM) containing EGTA (1 mM), mannitol (210 mM) and sucrose (70 mM) and adjusted to a pH of 7.2 (10 ml/g). Muscle aliquots were homogenised in buffer, using a glass on glass homoge- niser, and centrifuged at 1,500×g for 5 min at 4°C to remove insoluble connective tissue. For the detection of SOD1 and SOD2, cytosolic and mitochondrial fractions were separated. The supernatant was centrifuged at 10,000×g for 5 min at 4°C, and the resulting supernatant, containing the cytosolic fraction, was collected for SOD1 enzyme analysis. The remaining pellet containing the mitochon- drial fraction was resuspended and homogenised in ice- cold HEPES buffer (20 mM) for SOD2 enzyme analysis. SOD1 and SOD2 activity was measured in cytosolic and mitochondrial muscle fractions, respectively. The amount of enzyme activity and hydrogen peroxide levels were calculated and standardised for protein using the Bradford method (Bio-Rad).