The electron microscopy was conducted at the Shanghai Jiao Tong University Instrumental Analysis Center. Briefly, the liver tissues from Cp-LKO and littermate controls were excised into pieces of 1 mm3 and fixed overnight in 2.5% glutaraldehyde at room temperature. The samples were post-fixed in potassium ferrocyanide-reduced osmium tetroxide (Electron Microscopy Sciences, USA) for 1.5 h and dehydrated in a bath with gradually increasing concentrations of ethanol (50%, 70%, 90%). The ethanol was then eliminated with gradually increasing acetone concentrations (90% ethanol: 90% acetone = 1:1, 90% acetone, 100% acetone). Further, the tissues were incubated with a mixture of acetone: EPON (liquid epoxy resin) (1:1,1:2, and 1:3) and ultimately embedded with pure EPON (Electron Microscopy Sciences, USA). The picture was captured using a biological transmission electron microscope (Tecnai G2 Spirit BioTWIN, Thermo Fisher Scientific, USA). The liver tissues from the BCS-treated Cp-LKO mice and the control group were analyzed following the same procedures.
Potassium ferrocyanide reduced osmium tetroxide
Manufactured by Electron Microscopy Sciences
Sourced in United States
Potassium ferrocyanide-reduced osmium tetroxide is a chemical compound used in electron microscopy as a staining agent. It is a combination of potassium ferrocyanide and osmium tetroxide, which together enhance the contrast and visibility of cellular structures when preparing samples for examination under an electron microscope.
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3 protocols using potassium ferrocyanide reduced osmium tetroxide
1
Ultrastructural Analysis of Liver Tissues in Cp-LKO Mice
2
Ultrastructural Imaging of Bacterial Cells
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Some bacteria were fixed as above, post-fixed with potassium ferrocyanide-reduced osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) and then processed for embedding in Epon resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections of 80–100 nm were cut with a diamond knife on an Ultracut EM UC6 ultramicrotome (Leica Biosystems, Concord, ON, Canada) and transferred onto Formvar-coated 200-mesh nickel grids for TEM imaging. The grid-mounted sections were examined in a Tecnai 12 TEM (FEI (now Thermo Fisher Scientific), Eindhoven, Netherlands) operating at 80 kV. Some embedded samples were used tomographic imaging using a Crossbeam 550 Focused Ion Beam (FIB)-SEM (Carl Zeiss Microscopy, Oberkochen, Germany). Samples were serially milled at 4 nm thickness using a probe current of 1.5 nA/30 kV. Exposed surfaces were observed at 1.5 kV using Secondary Electron and Energy Selective Backscatter detectors. The Dragonfly software 2020.1 (Object Research Systems, Montréal, Canada) was used for further alignment and 3D reconstruction of the FIB-SEM image stacks.
3
Ultrastructural Analysis of Liver Tissues in Cp-LKO Mice
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The electron microscopy was conducted at the Shanghai Jiao Tong University Instrumental Analysis Center. Briefly, the liver tissues from Cp-LKO and littermate controls were excised into pieces of 1 mm3 and fixed overnight in 2.5% glutaraldehyde at room temperature. The samples were post-fixed in potassium ferrocyanide-reduced osmium tetroxide (Electron Microscopy Sciences, USA) for 1.5 h and dehydrated in a bath with gradually increasing concentrations of ethanol (50%, 70%, 90%). The ethanol was then eliminated with gradually increasing acetone concentrations (90% ethanol: 90% acetone = 1:1, 90% acetone, 100% acetone). Further, the tissues were incubated with a mixture of acetone: EPON (liquid epoxy resin) (1:1,1:2, and 1:3) and ultimately embedded with pure EPON (Electron Microscopy Sciences, USA). The picture was captured using a biological transmission electron microscope (Tecnai G2 Spirit BioTWIN, Thermo Fisher Scientific, USA). The liver tissues from the BCS-treated Cp-LKO mice and the control group were analyzed following the same procedures.
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