In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

Open Access
Publisher Website
Google Scholar
Abstract
In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. Two methods were developed to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The 1st method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on EM grids and fixed in 70% ethanol vapor; hybridization sites are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Interphase nuclei present in some of the metaphase chromosome preparations also show defined patterns of satellite DNA labeling which suggests that satellite-containing regions are associated with each other during interphase. The sensitivity of this method may be at least as good as that at the light microscope level while the resolution is improved at least 3-fold. The 2nd method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of colloidal gold particles (.apprx. 20 nm in diameter). Gold particles bind specifically both directly over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average 10 times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructural localization of DNA sequences in chromosomes and chromatin.