Ultrastructural immunolocalization of polyamines in HeLa cells subjected to fast-freezing fixation and freeze substitution

Abstract

Polyamines have been localized at the ultrastructural level in HeLa cells subjected first to fast-freezing fixation (FFF)-freeze substitution (FS) and then to an immunocytochemical method combining anti-polyamine antibodies and immunogold labelling. Polyamines were found in both the cytoplasm and the nucleus and, in the latter, particularly over the dense chromatin area. To our knowledge, this is the first example of the electron microscopic localization of a hapten after FFF-FS. For the preservation of fine ultrastructural details, this FFF-FS method is not only adequate but also greatly reduces, if not totally eliminates, any leeching-out and redistribution of the polyamines during the preparation of the sample. For the preservation of antigenicity in situ during FS, epoxy resin was more effective than hydrophilic LR white resin, probably due to the solubility of polyamines in the latter.

Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

“…1a). Also the lowered contrast is in agreement with several works mentioning lower contrast after using acrylic resins (Nicolas and Bassot 1993;Roch et al 1997;von Schack et al 1991von Schack et al , 1993Monaghan et al 1998). This feature, however, can be sometimes an advantage since gold particles become better visible over the cellular structures.…”

“…It has been also successfully used as a cell support for slam-freezing of HeLa cells (Roch et al 1997), and it appears that the combination of dense cell suspension and agarose in our protocol serves together well.…”

Profilin revealed in pollen nuclei: immuno-electron microscopy of high-pressure frozen Ledebouria socialis Roth (Hyacinthaceae)

“…One must be aware of potential pitfalls with immunolocalization of small macromolecules which have been stabilized by cryosubstitution with pure solvents (Schwarz and Humbel 1989;Roch et al 1997), particularly for specimens embedded in acrylics, which do not bind covalently to the tissue components; thus, insufficiently fixed low molecular weight antigens may become solubilized and, consequently, artifactually redistributed over the section by the water of the knife trough or the immuno-incubation media. In L. socialis anthers, however, the antigens under investigation apparently did not smear, as the labelling was confined strictly to pollen cytosol and nuclei.…”

A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles via a Two-step Mechanism

“…Attempts to determine the intracellular distribution of polyamines have relied on invasive techniques such as immunohistochemistry (10 -12) or subcellular fractionation (13). Whereas some have reported the presence of polyamines in both cytoplasm and nucleus (12,13), others have found an almost exclusively cytoplasmic distribution (10,11). One problem inherent to such techniques is the redistribution of polyamines that can rapidly follow fixation or membrane disruption, because of their high positive charge (1).…”