In Situ Hybridization Continued
      

Detection and Amplification. Factors such as tissue fixation, endogenous biotin or enzyme activity, desired sensitivity, and permanency of record should be considered when choosing either the optimal probe label or subsequent detection system. Protocols for fluorescence and enzyme based in situ detection are available in our brochure entitled, “A Guide to Nucleic Acid Labeling and Detection” (also available online). Various options are available for fluorescent or chromogenic visualization of in situ probes. When choosing the appropriate reagents, it is important to consider: 1. The label on the probe (e.g. biotin, fluorescein, etc.) 2. The visualization method (fluorescence or chromogenic detection) 3. The level of sensitivity required. Several streptavidin and avidin reagents, as well as antibodies to labels, are available directly conjugated to the detection moiety (fluorochrome or enzyme). These “one-step” reagents are convenient and sensitive enough for many applications. If additional sensitivity or flexibility is needed, many of the antibodies to labels can be detected with additional layers of amplification. For streptavidin- and avidin-based systems, use Biotinylated Anti-Streptavidin or Biotinylated Anti-Avidin reagents to amplify signal intensity in FISH applications. For non-biotin/streptavidin or non-biotin/avidin strategies, affinity-purified secondary antibody conjugates can be used for signal amplification. Associated Reagents. Increasing the accessibility of the labeled probe and detection reagents to the target sequence enhances sensitivity and specificity of an ISH signal. This can be achieved by reducing the size of DNA probes and pretreating tissues with a digestive enzyme. NicKit™ p.s.o. (MB-1905) has been developed for reducing the size of DNA probes to an optimal length for ISH without the problem of over- or under- digestion encountered with other methods. Proteinase K (VP-Y179) is used to digest proteins on tissue sections in order to enhance probe and detection reagent accessibility to the target. This enzyme unmasks nucleic acids from associated proteins. This step is often required when tissues have been treated with crosslinking fixatives like formaldehyde or glutaraldehyde. A protein blocking reagent is imperative to reduce background when detecting hybridized ISH probes. 5x In Situ Blocking Solution (MB-1220) is designed to be used as a general blocking agent and diluent for fluorescent and enzyme-based ISH.