208 and 0.2 mg for palmitoyl-CoA plus carnitine) were incubated in a medium consisting of 100 mM sucrose, 20 mM K+-Tes (pH 7.2), 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, 4 mM KH2PO4, 3 mM malate and 0.1% of FA-free BSA. Maximal coupled (state 3) respiration was initiated by addition of 450 M of adenosine diphosphate (ADP). State 4o respiration, reXecting proton leak, was measured as the residual respiration following addition of 1 g/ml oligomycin (an F1-F0 ATPase inhibitor). Maximal oxygen Xux rates (state uncoupled, state U) were obtained by titration of the chemical uncoupler car- bonyl cyanide p-triXuoromethoxyphenylhydrazone (FCCP). Addition of excess cytochrome C to the chamber in the pres- ence of pyruvate and ADP did not increase respiration (data not shown); indicating that during isolation outer mitochon- drial membrane integrity was retained. 14C-palmitate oxidation assay Ex vivo fat oxidation was measured as described (Frisard et al. 2010). BrieXy, muscle was homogenised in a buVer (pH 7.4) containing 250 mM sucrose, 10 mM Tris–HCl, 2 mM ATP and 1 mM EDTA (Sigma, St. Louis, USA). Subsequently, muscle homogenates were incubated for 2 h with [1-14C]-labelled palmitate (1 Ci/ml, Perkin Elmer, Boston, USA) and 200 M non-labelled (cold) palmitate in reaction media containing L-carnitine, ATP and coenzyme A (all from Sigma, St. Louis, USA) at 37°C. Palmitate was coupled to FA-free BSA in a molar ratio of 5:1. After incu- bation, reactions were terminated by adding 40 l of 70% perchloric acid, and the CO2 produced during the incuba- tion was trapped in 200 l of 1 N sodium hydroxide that had been added to adjacent wells (Kim et al. 2002). The acidiWed medium was stored at 4°C overnight, and then acid soluble metabolites (ASMs) were assayed in superna- tants of the acid precipitate. Radioactivity of CO2 and ASMs was determined by liquid scintillation counting by use of 5 ml of Hionic Fluor (Perkin Elmer, Boston, USA). Data were normalised to protein content. Electron microscopy procedures Ultrastructural morphology and PLIN5 localization were examined using transmission electron microscopy. For determinations of ultrastructural morphology, muscle tissue sections were Wxed in 2.5% glutaraldehyde in 0.1 M phos- phate buVer (pH 7.4). PostWxation was performed in 1% osmium tetroxide in 0.1 M cacodylate buVer (pH 7.4) sup- plemented with 1.5% potassium ferrocyanide. The samples were then dehydrated and embedded in epon. Ultrathin sections were examined using a Philips CM100 electron microscope. For determinations of subcellular localization of PLIN5, soleus, extensor digitorum longus (EDL) and heart muscle 123 Histochem Cell Biol (2012) 137:205–216 tissues 14-week old ZDF rats (ZDF/Gmi, fa/fa) and tibialis anterior muscle tissues from electroporated Wistar rats were cut into pieces with a maximum size of 1 mm3, Wxed for 1 h at room temperature in a mixture of 2% formalde- hyde and 0.2% glutaraldehyde in 0.1 M phosphate buVer (pH 7.4), and stored in 1% formaldehyde in 0.1 M phos- phate buVer (pH 7.4). Tissue pieces were immersed in a 2.3 M sucrose solution, frozen in liquid nitrogen and ultra- thin cryo-sections were cut under liquid nitrogen, sections were transferred from the knife edge to a formvar and car- bon-coated grid in 2.3 M sucrose/1.8% methylcellulose (1:1), as described by Tokuyasu (1973,1980,1986) Sections were immunogold labelled as described by Tokuyasu (1986) using a PLIN5 antibody (#GP31; Guinea pig poly- clonal; Progen Biotechnik, Heidelberg, Germany) in a 1:100 dilution and protein A-gold (20 nm particles) (Slot and Geuze 1981). Guinea pig normal serum (Santa Cruz Biotechnology, Tebu-bio, Heerhugowaard, The Nether- lands) was used as a negative control. Oil-red-O stainings and immunoXuorescence Neutral lipids, mitochondria and PLIN5 in muscle sections were stained with a modiWed Oil-red-O staining for Xuores- cence microscopy as previously described (Koopman et al. 2001) using antibodies against PLIN5 (#GP31; Progen Bio- technik, Heidelberg, Germany), laminin (L-9,393; Sigma, St. Louis, USA), and OXPHOS (MS601; MitoSciences, Eugene, OR, USA) and the appropriate secondary Alexa- Xuor conjugated antibodies (Invitrogen, Groningen, The Netherlands). Fluorescent images of Alexa-350 (laminin), Alexa-488 (PLIN5), Alexa-647 (OXPHOS), and Oil-red-O (visible in the red-594-channel) were acquired using an imaging system consisting of an IX81 inverted microscope (Olympus, Hamburg, Germany), an X-cite Xuorescent light source (Lumen Dynamics group, Mississauga, Ontario, Canada), F-view II CCD camera (OSIS, Münster, Germany) and the Cell^F imaging software (OSIS). Fluo- rescent photomicrographs were made from Z-stacks con- sisting of 30-40 images with a distance of 0.28 m using the maximum intensity projection method in the ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info. nih.gov/ij/, 1997–2009). Western blots Western blots were performed in protein lysates from whole muscle homogenates or mitochondrial fractions. Muscle samples and mitochondrial fractions were homoge- nised in ice-cold PBS containing 1% Nonidet-P40, 0.5% sodium dodecyl sulphate, 0.1 mM phenylmethylsulfonyl Xuoride, complete inhibitor (Roche, Almere, The Netherlands)