Electron microscopy in situ hybridization: tracking of DNA and RNA sequences at high resolution.
Electron microscopy in situ hybridization (EM-ISH) represents a powerful method that enables the localization of specific sequences of nucleic acids at high resolution. We provide here an overview of three different nonisotopic EM-ISH approaches that allow the visualization of nucleic acid sequences in cells. A comparison of various methods with respect to their sensitivity and the structural preservation of the sample is presented, with the aim of helping the reader to choose a convenient hybridization procedure. The post-embedding EM-ISH protocol that currently represents the most widely used technique is described in detail, with a special emphasis on the organization of the cell nucleus.
Cmarko
Dusan
D
Institute of Cellular Biology and Pathology, Ist Faculty of Medicine, Charles University, Prague, Czech Republic.
Koberna
Karel
K
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D020285:Cryoelectron Microscopy / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000648:ultrastructure*; D003850:Deoxyribonuclease I; D019286:Endopeptidase K; D005404:Fixatives; D020459:Genes, rRNA; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D008867:Microtomy / Q000379:methods; D015335:Molecular Probes; D009691:Nucleic Acid Denaturation; D016613:Plastic Embedding; D012313:RNA / Q000235:genetics* / Q000648:ultrastructure*; D012367:RNA, Viral / Q000235:genetics / Q000648:ultrastructure; D012259:Ribonuclease, Pancreatic
D005404:Fixatives; D015335:Molecular Probes; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic; D019286:Endopeptidase K