Uranyl acetate replacement stain

Specimens were fixed in fixation solution (0.12M PB buffer pH 7.4 containing 1% glutaraldehyde, 1% formaldehyde with 2mM calcium chloride and 2% sucrose) on ice for 1 hour and washed 5 x 3 minutes in cold 0.15M cacodylate buffer (50mM cacodylate, 50mM KCl, 2.5mM MgCl₂, 2mM CaCl₂ pH 7.4) on ice. After washing for 4 x 5 minutes in 0.15M cacodylate buffer at RT, specimens were subsequently fixed for 1 hr with 1% osmium tetroxide in 0.15M cacodylate buffer. Specimens were washed 2 x 5 minutes in ddH₂O. Afterwards, specimens were incubated for 1 hr in aqueous UAR-EMS (4%, Uranyl Acetate Replacement Stain, Electron Microscopy Sciences, Hatfield, USA), washed 2 x 5 minutes in ddH₂O at RT and dehydrated in an ascending ethanol series using solutions of 30%, 50%, 70%, 90%, 96%, and 100% ethanol for 10 minutes each. They were incubated two times in propylene oxide (PO) for 30 minutes each before incubation in a mixture of PO and Epon812 (1:1) overnight. The following day, the Epon-PO mixture was substituted with pure Epon812 and samples were incubated for 2 hours in Epon812. Specimens were embedded in Epon812 and kept at 60°C for 48hrs.

To evaluate skeletal muscle fine sub-cellular ultrastructural features, CB1^+/+ and CB1^−/− skeletal muscle were minced in small pieces and fixed overnight (O.N.) in glutaraldehyde 2.5% (Electron Microscopy Science, Hatfield, PA, USA) in 0.1 M cacodylate buffer (pH 7.4) at 4 °C. Fixed samples were then rinsed with 0.1 M cacodylate buffer for at least 1 h, post-fixed with 1% osmium tetroxide (OsO4) in 0.1 M cacodylate buffer (Electron Microscopy Science, Hatfield, PA, USA), dehydrated in ethanol (Sigma-Aldrich, Milano, Italy, EU), and embedded in epoxy resin (Electron Microscopy Science, Hatfield, PA, USA). Ultrathin sections (60 nm), obtained using an UC6 ultramicrotome (Leica, Wetzlar, Germany, EU) equipped with a diamond knife (DiATOME US, Hatfield, PA, USA), were placed on copper grids (Electron Microscopy Science, Hatfield, PA, USA). Ultrathin sections were then treated with Uranyl Acetate Replacement stain (UAR-Electron Microscopy Science, Hatfield, PA, USA) and contrasted with lead citrate (Sigma-Aldrich, Milano, Italy, EU). Samples were studied using a 100 kV transmission electron microscope EM208S PHILIPS (FEI—Thermo Fisher, Waltham, MA, USA) equipped with the acquisition system Megaview III SIS camera (Olympus/EMSIS) and iTEM3/Radius software, version 2.1.