Amplification methods to increase the sensitivity of in situ hybridization: play card(s)

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.