Several different fluorescent or enzyme-based systems are used for detecting labeled nucleic acid probes. These options provide the researcher with flexibility in optimizing experimental systems to achieve highest sensitivity, to avoid potential problems such as endogenous biotin or enzyme activity, or to introduce multiple labels in a single experiment. Such factors as tissue fixation, endogenous biotin or enzyme activity, desired sensitivity, and permanency of record should all be considered when choosing both the optimal probe label and subsequent detection system. The 5’ EndTag™ and 3’ EndTag™ Systems, PHOTOPROBE® Biotin products, and FastTag® Systems all label nucleic acid probes suitable for in situ hybridization applications. Choosing a labeling system generally depends on the type of nucleic acid probe being used. The 5’ or 3’ EndTag™ Kits are recommended for oligonucleotide labeling with a variety of labels at either end of the probe. Larger probes can also be labeled at multiple sites with either PHOTOPROBE® Biotin which introduces biotin or the FastTag® system, which allows a choice of labels to be coupled. In order to increase sensitivity, large DNA probes labeled with the FastTag® kit or PHOTOPROBE® Biotin can be reduced in size with the NICKIT™ p.s.o. probe size optimization kit (Cat. No. MB-1905). Vector Laboratories offers a wide range of detection methods that encompass visualization preferences and sensitivity requirements when performing in situ hybridization. Outlined below are examples of the reagents that can be used to detect biotin and fluorescein (the most common labels) using fluorescent or chromogenic techniques. Fluorescence In Situ Hybridization (FISH) For detection of biotinylated probes, select the following reagents: 5x In Situ Hybridization Blocking Solution Fluorescein Avidin DCS, Cell Sorting Grade Biotinylated Anti-Avidin D VECTASHIELD Mounting Media for Fluorescence Protocol: FISHB.pdf (PDF) For the detection of fluorescein-labeled probes, select the following reagents: 5x In Situ Hybridization Blocking Solution Biotinylated Anti-Fluorescein Fluorescein Avidin DCS, Cell Sorting Grade VECTASHIELD Mounting Media for Fluorescence Protocol: FISHF.pdf (PDF) Chromogenic In Situ Hybridization For the detection of biotinylated probes, select the following reagents: 5x In Situ Hybridization Blocking Solution Streptavidin, Alkaline Phosphatase conjugated BCIP/NBT AP Substrate Kit, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium Levamisole Solution VectaMount Permanent Mounting Medium Protocol: ISHB.pdf (PDF) For the detection of fluorescein labeled probes, select the following reagents: 5x In Situ Hybridization Blocking Solution Alkaline Phosphatase Anti-Fluorescein BCIP/NBT AP Substrate Kit, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium Levamisole Solution VectaMount Permanent Mounting Medium Protocol: ISHF.pdf (PDF) Guidelines For In Situ Hybridization Detection Please see the links below for further information and guidelines on in situ hybridization detection. ISH: Fluorescence Detection ISH: Enzyme-Based Detection The information contained in these webpages is available in PDF format: ISH.pdf (PDF) A general protocol for amplification of FISH using biotinylated Anti-Avidin: BA0300.pdf (PDF)