N-ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes.
We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3P = S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxynucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.
Zoeller
R T
RT
Department of Biology, University of Massachusetts, Amherst, USA.
Fletcher
D L
DL
Butnariu
O
O
Lowry
C A
CA
Moore
F L
FL
D001923:Brain Chemistry; D005033:Ethylmaleimide / Q000494:pharmacology*; D017403:In Situ Hybridization / Q000379:methods*; D015341:Nucleic Acid Probes / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry; D018075:RNA, Complementary / Q000737:chemistry; D012333:RNA, Messenger / Q000302:isolation & purification; D013462:Sulfur Radioisotopes; D013873:Thionucleotides / Q000737:chemistry*; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics / Q000302:isolation & purification
D015341:Nucleic Acid Probes; D015345:Oligonucleotide Probes; D018075:RNA, Complementary; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; D013973:Thyrotropin-Releasing Hormone; D005033:Ethylmaleimide