An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compare d to ISH with radiolabeled probes. To detect rare mRNAs, we optimized sever al parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted t yramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based, visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA pr obes (up to 2.61 KB) readily permeated cryosections and yielded stronger hy bridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detect ed mRNA for mGluR4 a receptor, and transducin, a G-protein, both of which a re expressed at very low abundance and are believed to be involved in chemo sensory transduction. Because the effect of the tested parameters was simil ar for ISH on sections of brain and tongue, we believe that these methodolo gical improvements for detecting rare mRNAs may be broadly applicable to ot her tissues.