Methods to Enhance Signal Using Isotopic In Situ Hybridization

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Abstract
Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.