Optimization of In Situ Hybridization and Co-expression Analysis in the Central Nervous System

Formalin-fixed paraffin-embedded (FFPE) tissues represent a treasure trove of material on the many diverse diseases of the central nervous system. Recent advances in the areas of in situ hybridization and co-expression analysis now allow for the routine detection of DNA, mRNA, microRNA, viral RNA, and proteins in these samples. Locked nucleic acid (LNA) probes have revolutionized the field of in situ hybridization by increasing the melting temperature of the DNA–DNA or cDNA/mRNA/microRA hybrids such that the probe only needs to have 20 nucleotides to achieve a strong signal and low background, even for low copy number targets. Both radioactive and colorimetric tagged LNA probes yield excellent sensitivity/specificity with good subcellular localization. It is important to stress that successful detection of the target depends on optimal tissue pretreatment. Thus, a simple optimization protocol for both immunohistochemistry and in situ hybridization using only two slides allows for simultaneous maximization of the signal to background ratio and determination of which targets can be successfully detected by co-expression analysis. This chapter will delineate the theory behind the recent advancements in this field and provide simple protocols based on the in situ detection of a variety of proteins, mRNAs, and microRNAs important in the pathophysiology of the brain.

Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news