PRETREATMENT IN PREEMBEDDING ELECTRON-MICROSCOPIC IN-SITU HYBRIDIZATION FOR DETECTION OF SPECIFIC RNA SEQUENCES IN CULTURED-CELLS - A METHODOLOGICAL STUDY

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in s itu hybridization (ISH). The specimens were monitored by reflection-co ntrast microscopy (RCM) before processing for EM. A good balance betwe en preservation of ultrastructure and intensity of hybridization signa ls was obtained by using mild aldehyde fixation followed by saponin pe rmeabilization, Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids we re detected immunocytochemically by the peroxidase/diaminobenzidine (D AB) method or by ultra-small gold with silver enhancement, Comparison of these methods favored the peroxidase/DAB system, The accessibility of RNA in the different cell compartments was dependent on the extent of crosslinking during primary fixation even after permeabilization wi th saponin, By using the most optimal ISH protocol and the peroxidase/ DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm b ut not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cyt oplasm, The sensitivity of the method is such that HEF housekeeping ge ne transcripts were detected in the cytoplasm.