Sensitive nonradioactive detection of mRNA in tissue sections: Novel application of the whole-mount in situ hybridization protocol

The relative insensitivity of nonradioactive mRNA detection in tissue secti ons compared to the sensitive nonradioactive detection of single-copy DNA s equences 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 resolutio n, we developed a nonradioactive in situ hybridization (ISH) protocol for d etection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increas e of the hybridization temperature to 70C while maintaining stringency of h ybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventiona l radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hyb ridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identific ation and localization of the cells in the developing heart that express lo w-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH pr ocedure scarcely permitted detection of these sequences, underscoring the v alue of this novel method.