ABSTRACT
In situ hybridization is one of the methods by which messenger RNA can be detected a specific messenger ribonucleic acid (mRNA) is detected by allowing a probe, i.e., a short, complementary DNA or RNA strand, to bind to the mRNA. This chapter introduces a sensitive and a very low-background assay to detect mRNA using in situ hybridization. It utilises in situ hybridization to detect changes in amounts of mRNA of some genes that might be affected by sleep deprivation. The brain sections are cut from frozen tissue; no fixation is needed before sectioning. Excessive use of fixative will give high background in dark field microscopy. Mild postfixation is needed to fix the macromolecules of the tissue on the slide. To reduce unspecific binding of the probe, the positively charged molecular groups in the tissue are acetylated by acetic anhydride. The chapter discusses the use of oligonucleotide probes.
