In situ hybridization (ISH) takes advantage of the ability of mRNA within a cell to hybridize with exogenously applied complementary RNA (riboprobes) or DNA molecules. This interaction is visualized by labeling the applied nucleic acid probe with a detectable molecule (radioactive, such as 35S, or nonradioactive, such as digoxygenin [DIG]). The technique allows patterns of gene expression to be visualized in many tissues or cell types simultaneously.
Nonradioactive ISH has several advantages over radioactive ISH. It lacks the biohazards associated with the use of radioisotopes, it takes days rather than weeks to get a visible signal, and it is cheaper. Using nonradioactive detection the degree of cellular resolution is significantly improved over that achieved with autoradiography. Another advantage of nonradioactive ISH is that it can be performed in combination with other assays with relative ease, in order to compare mRNA and protein distribution in the same tissues (1) as described in this chapter.
The procedures presented here are for ISH of DIG-labeled riboprobes to tissue sections from wax embedded tissues (methods for the use of frozen sections are generally similar but minor modifications may be required [2,3]). The use of tissue sections rather than whole mount embryos is preferable where the size of the embryo or the density of tissues leads to incomplete penetration of probe in whole mounts. The use of sections also allows a number of probes (for mRNA or proteins) to be used on the same or adjacent sections, an approach that is less straightforward in whole mounts. Both whole mount and section in situ hybridization protocols can therefore be used in a complementary way to give optimum results.
The procedures involved are:
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