Increased concentrations of radioisotopically-labeled complementary ribonucleic acid probe, dextran sulfate, and dithiothreitol in the hybridization buffer can improve results of in situ hybridization histochemistry

Abstract

The goal of the present studies was to optimize mRNA detection with radioisotopic in situ hybridization histochemistry (ISHH). Test experiments performed on sections of rat brain tissue used computer-assisted image analysis to compare autoradiographic signals resulting when varying concentrations of 35S-labeled cRNA probes, dextran sulfate (DS), and dithiothreitol (DTT) were used for ISHH. We found that greatly enhanced corrected signal density (total density of signal area minus background density) was obtained using concentrations of probe and/or DS that were several-fold higher than those widely recommended in published ISHH procedures (probe concentration >4 × 104 cpm/μl; DS concentration >10%). Extended hybridization reaction (>16 hr) also significantly augmented the corrected signal density. Finally, nonspecific probe binding was greatly reduced and corrected signal density enhanced by including 750-1000 mM, rather than the widely used 10-200 mM DTT, in the hybridization buffer. These observations indicate that the low efficiency of hybridization and the formation of high background may largely compromise the sensitivity of routine ISHH procedures. We suggest that the new method using increased concentrations of 3S-labeled cRNA probe, DS, and DTT will be especially important for the cellular localization of rare mRNA species.