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Journal of Histochemistry and Cytochemistry, Vol. 49, 1-8, January 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Sensitive Nonradioactive Detection of mRNA in Tissue Sections: Novel Application of the Whole-mount In Situ Hybridization Protocol

Antoon F. M. Moormana, Arjan C. Houwelinga, Piet A. J. de Boera, and Vincent M. Christoffelsa
a Experimental and Molecular Cardiology Group, Academic Medical Center, Amsterdam, The Netherlands

Correspondence to: Antoon F. M. Moorman, Dept. of Anatomy and Embryology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: a.f.moorman@amc.uva.nl

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method. (J Histochem Cytochem 49:1–8, 2001)

Key Words: nonradioactive in situ, hybridization, heart, development, Iroquois homeobox genes


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