In situ hybridization (ISH) is a technique for localization and detection of specific nucleic acid sequences within tissues and cells. DNA and RNA sequences are visualized by hybridization with labelled probes that are complementary to the sequence of interest. Hybridization histochemistry is a related term that refers specifically to RNA ISH.
When carrying out this technique, cells and tissue sections are typically fixed in 4% paraformaldehyde to preserve morphology for ISH. In some cases, tissues are permeabilized with proteinase K prior to hybridization to improve tissue penetration.
Probes are relatively short, labelled nucleotide sequences that are complementary to the sequence of interest. They are prepared by various enzymatic procedures with a reaction mixture that includes labelled nucleotide analogs or radioactive nucleotides, or by direct synthesis as an oligonucleotide. Probes may carry radioactive or fluorescent labels for direct detection, or hapten labels for detection by various indirect methods.
Once the sample has been prepared, it is incubated with the probe at elevated temperature to allow the probe to hybridize to the sequence of interest. Unhybridized probe is washed away and the remaining labelled probe is detected.
TSA and ISH
Tyramide Signal Amplification (TSA™) is a widely referenced technique for signal enhancement in ISH. It is ideal for challenging applications like detection of rare transcripts or detection of very short sequences like microRNA and may be used in any assay where it is possible to introduce horseradish peroxidase (HRP).
TSA requires a hapten labelled probe; typical probe labels include biotin, DNP, digoxigenin or fluorescein. Once the probe has been hybridized, HRP is introduced as a streptavidin or anti-hapten conjugate. HRP catalyzed deposition of the labelled TSA reagent immediate proximal the sequence of interest.
Visit the TSA references page to find out how scientists across the globe are using Tyramide Signal Amplification (TSA) for in situ hybridization techniques.
Detection of p53 mRNA in lung tissue. Digoxigenin-labelled p53 RNA probes were hybridized to paraffin n-embedded lung tissue. Comparison shows (A) standard digoxigenin detection with alkaline phosphatase/BCIP-NBT (60-minute substrate incubation) and (B) TSA Plus DNP with alkaline phosphatase/BCIP-NBT (15-minute substrate incubation).