In Situ Hybridization
In situ hybridization is a technique used to detect, visualize and localize DNA and RNA at the cellular level. Traditionally, detection of specific nucleic acid sequences localized in chromosomes, cells, or tissues has been via autoradiography using probes labeled with S35 or P33, and, more recently, chromogenic and fluorescence detection.
TSA offers a superior, nonradiometric alternative that takes less time, offers greater resolution, and enhances sensitivity versus radiometric techniques. When added to chromogenic ISH or FISH protocols, TSA produces amplification of signal that rivals sensitivity of radiometric ISH methods, while retaining excellent resolution.
A simple alternative to in situ PCR
Polymerase chain reaction (PCR) (Hoffmann-La Roche, Nutley, NJ) as been used with in situ hybridization (ISH) applications to boost assay sensitivity by amplifying the amount of target DNA present. This is advantageous when small amounts of starting DNA are available, but the procedure has drawbacks. It requires the use of special equipment, is tedious to carry out, sample contamination is fairly common, and resolution of the DNA or RNA signal may be poor.
Compared to PCR, TSA technology for in situ hybridization is readily added to existing protocols, and the procedure is quick and easy to perform. It requires no special equipment, and does not involve amplification and possible contamination of the target.
| Simpler and Faster Than IS-PCR, with Equivalent Sensitivity |
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| Detection of HIV-1 DNA in Formaldehyde-fixed Lymph Node Tissues from Positive Autopsy Material. |
Courtesy Dr. J. Luka and C. Afflerbach, Eastern Virginia Medical School, Norfolk, VA. |
| Sensitivity and Resolution Equivalent to Radiometric Methods, with Faster Results |
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| Detection of Enkephalin mRNA in the Rat Caudate. 33P or Digoxigenin-labeled enkephalin probe was hybridized overnight to a frozen rat brain tissue section. Detection of enkephalin mRNA was by 33P emulsion for two weeks (A), and TSA Plus (HRP) System |
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