A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.
The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.
Lan
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Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia.
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Nikolic-Paterson
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Atkins
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D000818:Animals; D006358:Hot Temperature; D017403:In Situ Hybridization / Q000379:methods*; D008872:Microwaves; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D012680:Sensitivity and Specificity; D016610:Tissue Embedding; D016707:Tissue Fixation
D012333:RNA, Messenger