A Complete Protocol for In Situ Hybridization of Messenger RNAs in Brain and Other Tissues With Radio-labeled Single-Stranded RNA Probes

Abstract

We present a detailed protocol for In Situ hybridization of messenger RNA molecules in brain and other tissues, using 35S-labeled single-stranded RNA transcribed from commercially available plasmid vectors. These vectors contain promoter sequences for specific synthesis of RNA, and polylinker regions that will accept cDNA inserts for virtually any target molecule of interest. The protocol is optimized for studies that include concomitant irnmunohistochemical and cytoarchitectonic evaluations, and it is sufficiently detailed for the molecular biologist unfamiliar with histologic technique or the histologist inexperienced in molecular biological methods. We have used it with consistant results for In Situ hybridization studies using probes to more than twenty different mRNAs; and for several of those we have obtained results showing quantitative differences in experimentally altered mRNA levels. Included are details of perfusion fixation, tissue sectioning and preparation, probe synthesis, hybridization reaction, and signal detection. The introduction and annotated comments include rationale for basic procedures and the purpose of various specific steps in the hybridization histochemistry protocol. (The J HistotechnoL 12:169, 1989).