Potassium ferrocyanide

Trypan Blue, Dulbecco’s Modified Eagle Medium (DMEM), penicillin (10,000 units), streptomycin (10 mg/mL), trypsin from porcine pancreas, 2-bromopalmitate (2-BP), palmitate, dimethyl sulfoxide (DMSO), potassium chloride, propidium iodide, RNase A, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), acridine orange, Giemsa stain, formaldehyde, paraformaldehyde, glutaraldehyde, Hoechst staining solution and poly-L-lysine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Transferrin-AlexaFluor 633, Albumin-AlexaFluor 488, rhodamine-123, goat anti-mouse IgG AlexaFluor 488 conjugate, goat anti-mouse IgG AlexaFluor 594 conjugate and Prolong Gold were purchased from Molecular Probes/Life Technologies (Eugene, OR, USA). Potassium ferrocyanide, Permount, sodium cacodylate, osmium tetroxide and PolyBed-812 resin were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Sodium dodecyl sulfate (SDS) was purchased from Ludwig Biotecnologia (Alvorada, RS, Brazil). Fetal bovine serum (FBS) was purchased from Gibco/Invitrogen/Life Technologies (Eugene, OR, USA). Nonidet 40 (NP-40) was purchased from Anresco Laboratories (San Francisco, CA, USA).

Liver samples were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, United States, cat. 16220), post-fixed with osmium tetroxide (Electron Microscopy Sciences, United States, cat. 19100) and potassium ferrocyanide (Electron Microscopy Sciences, United States, cat. 20150) for 60 min, dehydrated with increasing series of PA acetone (VETEC, BRAZIL, cat. 187) and infiltrated with EPOXI resin (EMbed812, Electron Microscopy Sciences, United States, cat. 14120). After polymerization, 1 µm semi-fine slices were made to choose the region to be analyzed. Then, 70 nm ultrathin sections were made; these were contrasted with 1% uranyl acetate (Electron Microscopy Sciences, United States, cat. 22400) and 1% lead citrate (Electron Microscopy Sciences, United States, cat. 17800), and visualized using Jeol transmission electron microscope JEM-1011 (JEOL Ltd., Akishima, Japan). Digital micrographs were captured using an ORIUS CCD digital camera (Gatan Inc., United States) at 5,000× magnification, and later a qualitative analysis was carried out. The mitochondria and endoplasmic reticulum integrity, amount of glycogen grains, and cytoplasm were evaluated in 15 fields per grid of each animal. (n = 5/group).