N-ETHYLMALEIMIDE (NEM) CAN SIGNIFICANTLY IMPROVE IN-SITU HYBRIDIZATION RESULTS USING S-35 LABELED OLIGODEOXYNUCLEOTIDE OR COMPLEMENTARY RNAPROBES

We predicted that a significant source of background labeling after in situ hybridization (ISH) using S-35-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 immobiliz e 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 d isulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of S-35-labeled oli godeoxynucleotide 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 s ignal-to-noise ratio.