InSitupedia

It is one of the most important steps. Proteinases (eg. proteinase K) digest the protein surrounding the target sequence which enhances the accessibility of the probe to allow proper hybridization.

Other enzymes, such as pepsin, are useful for formalin-fixed, paraffin embbeded tissues; collagenase and dispase are used in tissues prone to produce too much background reaction, such as connective tissue and liver. 

A prolonged incubation results in signal loss and affects tissue integrity. Incubation time and proteinase concentration must be optimized for the tissue being subjected to the protocol.

For probe addition, concentration must be well calculated beforehand. The concentration affects the rate of the first base pair formations between the target nucleic acid sequence and the probe and should be high enough for signal detection.

Probes are labeled with marker molecules (eg. DIG, biotin) in order to be visualized. This ISH signal detection is possible by performing histochemical or immunohistochemical methods that involve anti-label antibodies.

For more information on probes click here.

Antibodies detecting probe-target hybridization can be conjugated with reporter molecules. These include:

• Enzymes - antibodies are coupled with enzymes capable of generating coloured products (eg. Alkaline Phosphatase).

• Fluorochrome - fluorochrome-labeled antibodies need to be visualized by a fluorescent microscope that can detect the wavelenght emmited by fluorescent dyes.

• Colloidal gold - These antibodies are generally used in cryostatic sections visualized by electron microscopy.

The ISH signal is detected differently according to the type of technique used:

• Radioisotopic ISH is detected by x-ray film or by emulsive autoradiography.

• Nonisotopic ISH uses colorimetric or fluorescent substrats. 

For the enzyme alkaline phosphatase, for example, substrats used include NBT and BCIP (colorimetric) and HNPP (fluorescent).