The recommended protocol for in situ hybridization

Abstract Up until this chapter, none of this book has been a “cookbook” of how to use in situ hybridization or immunohistochemistry. I developed the book this way because of my strong belief that the stronger foundation you have in the theory of the methodologies, the better you will be able to develop your own protocol and troubleshooting strategy. This chapter presents my cookbook recipe to do in situ hybridization. It can serve as a starting point for you to develop your own protocol. The protocol in this chapter stresses several key points. They include the importance of using several different concentrations of the probe and varying the pretreatment conditions. This strategy will increase the likelihood that you will find the optimal conditions for the detection of RNA or DNA for any given tissue. Another key point stressed in this chapter is the importance of monitoring the in situ reaction so as to maximize signal and reduce background.