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Potential problems in using [35S]-dATP-tailed oligonucleotides for detecting mRNAs in certain cells of the immune
E Mezey1, B J Hoffman, G Harta
1Laboratory of Clinical Sciences, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland.
Abstract:
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.
Insights
Intense signals in immune cell studies using 35S-labeled probes are caused by signal amplification. This can be prevented by modifying probe synthesis or hybridization conditions.
Area of Science:
- Molecular Biology
- Immunohistochemistry
- Biochemistry
Background:
- In situ hybridization histochemistry (ISHH) using 35S-labeled oligonucleotides can produce unusually intense signals in immune cells.
- This phenomenon requires further investigation to understand its underlying mechanisms and implications in cellular research.
Purpose of the Study:
- To investigate the cause of intense signals observed in immune cells during ISHH with 35S-labeled oligonucleotides.
- To identify factors contributing to signal amplification and explore methods to mitigate this effect.
Main Methods:
- Examination of signal intensity in immune cells using 35S-labeled oligonucleotide probes in ISHH.
- Analysis of chemical interactions and the role of oxidative enzymes in signal amplification.
- Testing modifications in probe synthesis (e.g., MgCl2 vs. CoCl2, 33P vs. 35S labeling) and hybridization buffer (e.g., addition of 2,3-dimercaptopropanol [BAL]).
Main Results:
- The intense signals are a result of specific signal amplification due to chemical interactions after probe-mRNA binding.
- Oxidative enzymes present in immune cells (macrophages, neutrophils, eosinophils) are necessary for this amplification.
- Signal intensification can be avoided by substituting MgCl2 with CoCl2, adding BAL to the buffer, or using 33P-labeled probes.
Conclusions:
- Signal amplification in ISHH of immune cells is mediated by oxidative enzymes and specific chemical reactions.
- Optimized probe labeling and hybridization conditions can effectively control signal intensity, improving the reliability of ISHH studies.