The development, using poly(Hg-U) in a model system, of a new method to visualize cytochemical hybridization in fluorescence microscopy.
The development, using a model system, of a new method for the detection of cytochemical (in situ) hybrids is described. The method is based on the mercuration of nucleic acids with mercuric acetate. To facilitate hybridization, the acetate ligand is replaced by the CN- ion. In the hybrids formed, the CN- is exchanged for trinitrophenyl(TNP)-glutathione. The TNP-glutathione is subsequently detected by indirect immunofluorescence using anti-TNP antibodies. The feasibility of the approach was investigated using Sepharose- or Sephadex-bound poly(A) and mercurated poly(U). Poly(Hg-U) did hybridize with poly(A)-Sepharose, provided that the acetate ligand was replaced with CN-. The TNP-glutathione hapten thus synthesized bound effectively to mercury-Sepharose but not to amino-Sepharose when the reaction was performed in the dark. Furthermore, binding of TNP-glutathione to Sephadex-bound poly(A) . poly(HG-U) hybrids was detectable with indirect immunofluorescence using anti-TNP antibodies. The fluorescence intensity measured was dependent on the amount of poly(Hg-U) present and on the dilution of the antibody. Nonspecific binding was very low. Calibration of the number of fluorescein molecules found after the complete reaction was performed with fluorescein isothiocyanate-labeled poly(U). It was determined that one fluorochrome molecule per two nucleotides had been obtained, in close agreement with the theoretically expected number. The sensitivity of the method, when applicable to microscopic preparations, is comparable to in situ hybridization with 3H-labeled nucleic acids with a specific activity of 4 x 10(8) dpm/micrograms (two 3H-isotopes per nucleotide) and an exposure time of 1 day. Extension of the method to the cRNA-DNA system and its application to microscopic preparations is under investigation.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005455:Fluorescent Antibody Technique; D005978:Glutathione / Q000378:metabolism; D006241:Haptens / Q000032:analysis; D006651:Histocytochemistry; D008628:Mercury; D008856:Microscopy, Fluorescence / Q000379:methods*; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000235:genetics; D011072:Poly U / Q000235:genetics*; D012313:RNA / Q000235:genetics; D014301:Trinitrobenzenes / Q000378:metabolism
D006241:Haptens; D014301:Trinitrobenzenes; D011061:Poly A; D011072:Poly U; D012313:RNA; D008628:Mercury; D005978:Glutathione