To detect endothelial glycocalyx using electron microscopy [21 (link)], mice were anesthetized and perfused with a solution composed of 2% glutaraldehyde, 2% sucrose, 0.1 M sodium cacodylate buffer (pH 7.3), and 2% lanthanum nitrate through a cannula placed in the left ventricle 48 h after LPS administration [22 (link)]. Before perfusion, an incision was made in the right atrial appendage, and the neck was ligated with a silk suture. In addition, a perfusion pump was used for injection at a steady rate of 1 ml/minute.
Thereafter, the left ventricle, liver, and kidney were harvested and diced. Three or four pieces of approximately 1 mm3 each were immersed in the perfusion solution for 2 h for fixation and then soaked overnight in a solution without glutaraldehyde before being washed in alkaline (0.03 mol/L NaOH) sucrose (2%) solution. The specimens were then dehydrated through a graded ethanol series.
The frozen fracture method was used to prepare samples for examination using scanning electron microscopy (SEM). Each sample was laid on an iron plate chilled with liquid nitrogen, and ethanol was sprinkled onto it. Once the ethanol was frozen, the sample was fractured using a chisel such that it was not touched directly. The samples were then incubated in tert-butyl alcohol at room temperature. After the tert-butyl alcohol had solidified, it was freeze-dried, and the specimens were examined using SEM (S-4500; Hitachi, Tokyo, Japan). In addition, for further elemental analysis of each sample, energy-dispersive X-ray spectroscopy was performed under SEM.
To prepare samples for transmission electron microscopy (TEM), each specimen was embedded in epoxy resin. Ultrathin sections (90 nm) stained with uranyl acetate and lead citrate were then examined using TEM (HT-7700; Hitachi). For usual electron microscopy, 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) was used instead of perfusion buffer as described above.
Thereafter, the left ventricle, liver, and kidney were harvested and diced. Three or four pieces of approximately 1 mm3 each were immersed in the perfusion solution for 2 h for fixation and then soaked overnight in a solution without glutaraldehyde before being washed in alkaline (0.03 mol/L NaOH) sucrose (2%) solution. The specimens were then dehydrated through a graded ethanol series.
The frozen fracture method was used to prepare samples for examination using scanning electron microscopy (SEM). Each sample was laid on an iron plate chilled with liquid nitrogen, and ethanol was sprinkled onto it. Once the ethanol was frozen, the sample was fractured using a chisel such that it was not touched directly. The samples were then incubated in tert-butyl alcohol at room temperature. After the tert-butyl alcohol had solidified, it was freeze-dried, and the specimens were examined using SEM (S-4500; Hitachi, Tokyo, Japan). In addition, for further elemental analysis of each sample, energy-dispersive X-ray spectroscopy was performed under SEM.
To prepare samples for transmission electron microscopy (TEM), each specimen was embedded in epoxy resin. Ultrathin sections (90 nm) stained with uranyl acetate and lead citrate were then examined using TEM (HT-7700; Hitachi). For usual electron microscopy, 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) was used instead of perfusion buffer as described above.
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