Electron microscopic in situ hybridization in amphibian lampbrush chromosomes

Electron microscopy RNA/RNA in situ hybridization was adjusted to Pleurodeles lampbrush chromosomes. The cRNA probe used was synthesized from genomic DNA sequences which were proven to be moderately repetitive. Preembedding hybridization was performed on lampbrush chromosome preparations, and several fixation and hybridization conditions were tested. Paraformaldehyde and glutaraldehyde were used as fixatives at various concentrations and durations. Hybridization was then performed with or without formamide for various times of incubation. The best results were obtained when hybridization was carried out for 4 h at 42 degrees C in the presence of formamide, with lampbrush chromosomes having been previously fixed overnight in a mixture of paraformaldehyde/glutaraldehyde. Due to the excellent preservation of the ultrastructure and specificity of the signal obtained under these conditions, we were able to demonstrate, at the ultrastructural level, that such RNA expression occurs in two types of lampbrush loop ribonucleoprotein matrices showing a different morphology of their transcripts. Furthermore, a different mapping of the same sequence along the loop DNA axes is strongly suggested by a different labeling distribution in these loops.