CRYOFIXATION, CRYOSUBSTITUTION, CRYOEMBEDDING FOR VISUALIZING OF NUCLEAR ULTRASTRUCTURE AND FOR IMMUNODETECTION IN HELA-CELLS

We investigated the nuclear ultrastructure of HeLa cells using cryotechniques. The cell were cultured as monolayers, quick-frozen and cryosubstituted in acetone without chemical fixatives. Half the samples were then treated with 1% osmium tetroxide in acetone and embedded in Epon, protocol A. The other half was cryo-embedded in Lowicryl K11M at -60-degrees-C, protocol B. The efficiency of cryofixation was observed with both types of procedure. There was no fixation gradient detectable within the monolayer. The nuclei were well preserved, showing no reticulation of the chromatin. The different nuclear structures which have been observed after chemical fixation were also visible in these preparations. We noted improved visualization of the lamina and nucleolar chromatin, a very sharp limit between the fibrillar center and the dense fibrillar component, and variability in the appearance of the nuclear pores. Immunolabelling with an auto-immune serum was possible on sections processed by protocol A and protocol B, but not on sections obtained by chemical fixation and cryo-embedding procedures. Therefore, in the present work we showed that cryofixation, cryosubstitution and cryo-embedding of cell monolayers can ensure a good preservation of the nuclear structures. Furthermore, detection of nucleolar antigenic determinants is facilitated and rapid nuclear events can easily be monitored.