Ultrastructure of Kurloff body proteoglycans after high pressure freezing, cryosubstitution and postembedding staining with cuprolinic blue

Abstract

Very high pressure freezing and cryosubstitution of Kurloff cells preserves the ultrastructural morpholgy of Kurloff bodies, particularly the myelin figures, as shown by embedding in epoxy resin and conventional postembedding staining. It also preserves the Kurloff body proteoglcans as more expanded spindle-like shapes than does fixation wth formaldehyde at atmospheric pressure. But, proteoglycans were not discernible in the Kurloff body matrix on either unstained or conventionally stained thin sections. The Kurloff body skeleton of proteoglycans in their native expanded shape was stained with the electron-dense cationic ministain cuprolinic blue, using thin sections embedded in LR white. The mean equatorial diameter of the spindles was 20-30 nm, while the collapsed filaments produced by aldehyde fixation were about 10-15 nm wide. The spindles were often about 200-300 nm long but could be much longer, depending on the plane of the section. Thus, high pressure freezing, freeze substitution, embedding in LR white, and staining with cationic dyes such as phthalocyanins seems to be a convenient way of visualizing intracellular proteoglycans that are well preserved and in very much like their native expanded state.