( Using 1 g of gel-purifiedtemplate D...)The DIG System Nonradioactive Automated,,,High-Throughput In Situ Hybridization:,,,a Powerful Tool for Functional Genomics Research

Using 1 g of gel-purified template, DIG-labeled RNA probe is produced by in vitro transcription according to the instructions of the DIG RNA Labeling Kit (Roche Applied Science). The probe concentration is adjusted to 100 ng/l in hybridization buffer. DIG RNA probes are stored at -20C until used.

Tissue preparation

Adult mouse brain or 14.5-day-old embryos are isolated, placed in an embedding chamber containing O.C.T. 4583 (Tissue-Tek, Sakura) and slowly frozen. Tissues are sectioned using a Leica CM3050S cryostat to a thickness of 20 m, placed on Super Frost Plus microscope slides.

Sections are fixed for 20 minutes in a solution of 4% paraformaldehyde (PFA, EMS) in phosphatebuffered saline (PBS), washed, acetylated, and dehydrated through graded ethanol series. The slides can be stored in air-tight moisture-protected chambers at -80C for at least 3 months.

Prehybridization treatments

After adjusting to room temperature for several hours, slides are assembled in the flow-through chambers. To prepare the tissue sections for hybridization with the RNA probe, they are submitted to a series of prehybridization treatments. All required solutions are prepared and placed in heatable or ambient temperature reservoirs on the robot platform. The robot performs each step automatically by pipetting the solutions into the slide chambers according to the programmed script. A wide variety of scripts can be used, corresponding to the desired in situ hybridization protocol.

Hybridization

DIG-labeled RNA probes are denatured and placed into the appropriate positions in the heatable microreaction vial rack on the instrument platform. A minim
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