Backscattered electron imaging of crystal matrix protein on the surface of calcium oxalate crystals using colloidal gold.

Abstract

In order to clarify the presence and localization of crystal matrix protein (CMP) upon calcium oxalate crystals, scanning electron microscopy (SEM) and backscattered electron imaging (BEI) techniques were used. This protein exhibits a remarkable affinity with calcium oxalate crystals and may be important in stone pathogenesis. In this paper, rabbit anti-human CMP polyclonal antibody was used as first antibody, and for the second antibody, goat anti-rabbit IgG conjugated with 20 nm immunogold was used. Freshly prepared crystals from male urine were fixed in SEM fixative, then blocked and washed with phosphate-buffered saline and bovine serum albumin (PBS/BSA). First and second antibodies were reacted in PBS/BSA. Crystals were then dehydrated and finally coated for SEM study. The SEM technique showed bipyramidal shaped dihydrate calcium oxalate crystals in every sample and even at high magnification, colloidal gold could barely be seen. BEI clearly demonstrated the presence and localization of the gold on the surface of the crystals as well as on the macromolecules eluted from the crystals by dissolving them in ethylenediamminetetraacetic acid solution.