Potential problems in using [35S]-dATP-tailed oligonucleotides for detecting mRNAs in certain cells of the immune system.

In this study we examined the cause of unusually intense signals obtained in immune cells by in situ hybridization histochemistry using 35S-labeled oligonucleotides. We verified that the phenomenon is an amplification of a specific signal due to a series of chemical interactions after the probe binds to a specific mRNA in the tissue. The presence of oxidative enzymes in the tissue seems to be necessary for this reaction to occur. Therefore, most cells of the immune system (e.g., macrophages, neutrophil and eosinophil leukocytes), being rich in oxidative enzymes, will show some signal amplification. The intensification of the signal can be avoided if MgCl2 is substituted for CoCl2 in the synthesis of [35S]-thiophosphate-labeled probes, if 2,3-dimercaptopropanol [British anti-Lewisite (BAL)] is added to the hybridization buffer, or if [33P]-phosphate is used instead of [35S]-thiophosphate in the labeling of the probes.