Livers were perfused, cut into pieces, fixed and washed as described for scanning EM (see above).
For the analyses of tight junction functionality, mouse livers were additionally perfused through the inferior vena cava with 3% (w/v) lanthanum nitrate in 4% (w/v) PFA for 10 min28 (link) after short-term perfusion with 4% (w/v) PFA for 1 min.
The samples were prepared according to procedures described before70 (link). In brief, for contrasting, liver samples were incubated with 1% (w/v) OsO4 for 2 h and washed three times with 0.1 M sodium cacodylate buffer. Samples were then dehydrated by rising ethanol concentrations and stained with 2% (w/v) uranylacetate in 50% (v/v) ethanol for 1 h before they were embedded in araldite resin at 60 °C for 48 h.
After ultrathin sectioning of the embedded samples using a LKB 8800A Ultratome III (LKB Produkter AB), the sections (60 nm) were placed on formvar-coated grids and were finally stained with 3% (w/v) lead citrate in ddH2O (Electron Microscopy Sciences) for 2 min.
The sections were investigated in an EM902A transmission electron microscope (Carl Zeiss AG) operated at 80 kV and images were recorded with a 1 k FastScan CCD camera (TVIPS camera and software).
For the analyses of tight junction functionality, mouse livers were additionally perfused through the inferior vena cava with 3% (w/v) lanthanum nitrate in 4% (w/v) PFA for 10 min28 (link) after short-term perfusion with 4% (w/v) PFA for 1 min.
The samples were prepared according to procedures described before70 (link). In brief, for contrasting, liver samples were incubated with 1% (w/v) OsO4 for 2 h and washed three times with 0.1 M sodium cacodylate buffer. Samples were then dehydrated by rising ethanol concentrations and stained with 2% (w/v) uranylacetate in 50% (v/v) ethanol for 1 h before they were embedded in araldite resin at 60 °C for 48 h.
After ultrathin sectioning of the embedded samples using a LKB 8800A Ultratome III (LKB Produkter AB), the sections (60 nm) were placed on formvar-coated grids and were finally stained with 3% (w/v) lead citrate in ddH2O (Electron Microscopy Sciences) for 2 min.
The sections were investigated in an EM902A transmission electron microscope (Carl Zeiss AG) operated at 80 kV and images were recorded with a 1 k FastScan CCD camera (TVIPS camera and software).
Free full text: Click here
