In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.

We have developed an easy, stream-lined yet sensitive protocol for in situ hybridization to mRNA in frozen tissue sections or cytospins using digoxigenin-labeled RNA probes detected by alkaline phosphatase. We found the crucial parameters for successfully performing this technique to be tissue quality, fixation time and effective removal of excess probe. Most preparation, incubation steps and washes as described in the literature were found to be unnecessary and, therefore, eliminated, making this protocol simple enough to be achieved in any laboratory with access to tissue preparation and some molecular biology expertise.