A non-radioactive in situ hybridization method that does not require RNAse-free conditions

This report describes a quick and versatile method to perform non-radioactive in situ hybridization in which none of the hybridization steps are performed under RNAse-free conditions. This study demonstrates that in situ hybridization can be performed without an RNAse-free environment provided that the concentration of RNAse introduced during the experiment does not reach 0.1 microg/ml, a concentration that is unlikely to be achieved through an accidental contamination. Moreover, evidence is provided that the only step sensitive to RNAse degradation is the pretreatment since degradation during the hybridization step can not occur due to a very efficient protective effect exerted by formamide. Finally, our data suggest that endogenous RNAse activity might be readily neutralized through paraformaldehyde fixation. A feature of this method is the strong fixation that ensures a perfect tissue preservation, even at level of the fine structure of the cell processes. The method allows a uniform tissue penetration by sodium periodate and sodium borohydride treatment and can be easily used in combination with diaminobenzidine immunohistochemistry for double labeling experiments.