Osmium tetroxide oso4

Shortly after dissection, liver tissue was cut in small pieces (1–2 mm) and fixed in 3% glutaraldehyde in 1× PBS. Samples were washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), post-fixed in 1% Osmium tetroxide (OsO₄) (Roth, Karlsruhe, Germany) in 17% sucrose buffer (Merck) and dehydrated by ascending ethanol series (30%, 50%, 70%, 90% and 100%) for 10 min each. The last step was repeated 3 times. Dehydrated specimens were incubated in propylene oxide (Serva) for 30 min, in a mixture of Epon resin (Serva) and propylene oxide (1:1) for 1 h and finally in pure Epon for 1 h. Samples were embedded in pure Epon. Epon polymerization was performed at 90 °C for 2 h. Ultrathin sections (70–100 nm) were cut with an ultramicrotome (Reichert Ultracut S, Leica, Wetzlar, Germany) using a diamond knife (Diatome Ltd., Nidau, Switzerland) and picked up on Cu/Rh g (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany). Contrast was enhanced by staining with 0.5% uranyl acetate and 1% lead citrate (both from EMS GmbH, Munich, Germany). Samples were viewed at an acceleration voltage of 60 kV using a Zeiss Leo 906 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope. Pictures were acquired in magnifications of 6000×, 10,000×, 27,000× and 60,000×.

Electron microscopic analysis of Atp7b^−/− mouse liver tissue was done essentially as described before [31 (link)]. In brief, after dissection of the livers, small tissue samples with a size 1–2 mm were prepared and fixed in 3% glutaraldehyde prepared in 1× phosphate buffered saline (PBS). Samples were washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), post-fixed in 1% Osmium tetroxide (OsO₄) (Roth, Karlsruhe, Germany) in 17% sucrose buffer (Merck), dehydrated through ascending ethanol series, and embedded in epoxy resin as reported before [31 (link)]. Ultrathin sections (70–100 nm) were prepared and spread on Cu/Rh grids (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany). The samples were examined with a Zeiss Leo 906 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope operated at 60 kV.