A RIBONUCLEASE-RESISTANT METHOD OF IN-SITU HYBRIDIZATION HISTOCHEMISTRY IN RAT-BRAIN TISSUE

Two major problems limiting neurobiological applications of in situ hy bridization are: (1) contamination by ribonuclease (RNase), which is d ifficult to avoid and therefore makes the method difficult to establis h for many laboratories, and (2) lack of reproducibility, which makes the method inadequate for detecting and quantifying changes in mRNA le vels. We have developed a modified method of in situ hybridization whi ch addresses these problems. RNase resistance is afforded by the inclu sion of RNase inhibitors during steps in which mRNA is vulnerable to R Nase digestion, alleviating the need to maintain RNase-free conditions during experiments. These changes result in higher levels of specific hybridization, while maintaining low background. In addition, a high level of reproducibility is obtained, both for sections obtained from the same animal and for corresponding sections obtained from different animals. This method has been characterized for preproenkephalin and glutamate receptor GluR1-4 mRNAs.