Silver-enhanced immunogold scanning electron microscopy using vibratome sections of rat kidneys: detection of albumin filtration and reabsorption

Abstract

To show the three-dimensional distribution of proteins in renal cells, we applied the immunogold scanning electron microscopy method using vibratome slices. Kidney specimens from puromycin aminonucleoside (PAN) nephrotic rats and controls were obtained after intravenous infusion of human serum albumin and fixed in periodate-lysine-paraformaldehyde solution. Vibratome slices were incubated with anti-human albumin antibody and 25-nm gold-labeled secondary antibody. After silver enhancement, the immunogold particles were clearly observed by backscatter electron imaging, whereas they were ambiguous in the secondary electron image. The images showed a higher resolution of the tissues at an acceleration voltage of 5 mV than at 10 mV. Immunogold-labeled albumin was observed in the lumen and endocytotic vesicles of the proximal tubules, and on the podocyte surface in the PAN nephrotic rats, whereas only a few particles were observed in the controls. In conclusion, silver-enhanced immunogold scanning electron microscopy at low acceleration voltages using vibratome sections can be applicable for detecting the intracellular/extracellular localization of molecules in solid tissues. We succeeded in visualizing the enhanced albumin endocytosis of the proximal tubules and the exocytosis of albumin from podocytes in the nephrotic rats.