Molecular hybridization of RNA and DNA in situ: visualization at the electron microscope level

Abstract

SUMMARY Electron microscopic visualization of molecular hybrids formed in situ is feasible at the present time. It can be accomplished by two alternative approaches. In one, the in situ hybridization is carried out on ultrathin sections of target embedded in glycol methacrylate. In the other, whole cells are used for hybridization and they are subsequently prepared for electron microscopy. The choice of the method to be adopted depends on the type of target tissue. When there is a choice, the second approach seems preferable. Some of the important technical steps in the hybridization procedure, such as DNA denaturation in ultrathin sections, have been discussed and attention has been drawn to practical problems that may arise during the preparatory steps. Our light microscope experiments demonstrate that preparations made after glutaraldehyde fixation have a lower hybridization efficiency than those fixed with 3: 1 methanol‐acetic acid. Attempts are therefore being made to explore the possibility of using methanol‐acetic acid for electron microscope in situ hybridization. First results of straightforward fixation show that the preservation of nuclear structure may be fairly satisfactory for the purpose. However, the cumulative effects of subsequent treatments in the procedure still remain to be examined. For electron microscope autoradiographs (EM ARG) of hybridized preparations, the most suitable emulsion at present appears to be Ilford L4. Various factors conducive to optimum resolution consistent with maximum efficiency in this emulsion have been pointed out. Practical problems that may arise in autoradio graphs of hybridized preparations such as background and variation of grain density in adjacent sections have also been considered.