( um of 120 l(optimal are 300 l) probe...)The DIG System Nonradioactive Automated,,,High-Throughput In Situ Hybridization:,,,a Powerful Tool for Functional Genomics Research

um of 120 l (optimal are 300 l) probe is added per slide, and hybridization is carried out at 60C overnight. Within the range of 10 80C, the temperature varies only by 0.5C in each individual flow-through chamber. The temperature variation across the whole thermorack is 1.0C. This highly accurate temperature control ensures consistent and reproducible results within one experiment, and between different experiments.

Posthybridization treatments

After hybridization, stringency washes are carried out at the desired temperature (typically 60C) to remove unbound RNA probes. The robot pipettes the preheated washing solutions (SSC/formamide) from their heatable reservoirs on the platform into the heated flow-through chambers, so that there is no loss of temperature or bubble formation on the slides.

Antibody-mediated detection

At ambient temperature, several blocking steps are carried out to reduce nonspecific background. After the blocking steps, antidigoxigenin antibody is applied to the slides. Typically, these antibodies consist of Fab fragments that are linked to alkaline phosphatase, peroxidase, or another enzyme for colorimetric detection (Roche Applied Science). An optional signal amplification step (TSA system, Perkin Elmer Lifesciences) helps to detect transcripts of weakly expressed genes.

After several washing steps to remove unbound antibody, the substrate for color reaction (BCIP and NBT, Roche Applied Science) is applied and slides are incubated until the desired signal intensity is reached. The reaction can be timed manually or programmed to a specific time. The reaction is then stopped and the color precipitate is fixed for slide mounting and microscopy. Slides are left to dry overnight an
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