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The DIG System Nonradioactive Automated,,,High-Throughput In Situ Hybridization:,,,a Powerful Tool for Functional Genomics Research

arvae, or tiny plant root tips, instruments optimized for whole-mount in situ hybridization (e.g., Intavis) are suitable. However, the large majority of tissues are too large for the whole-mount method and have to be dissected, sectioned, and attached to microscope slides for further analysis.

The Max Planck Institute of Experimental Endocrinology in Hanover and the Swiss lab automation company Tecan have jointly developed a new system to process microscope slides with tissue sections. The slides carrying tissue sections are assembled into flow-through chambers and remain there until the end of the procedure. Capillary forces ensure that the tissue sections are liquid-covered at all times and protected from damage and desiccation. All solutions are added to the chambers by the computer-controlled liquid-handling system and displace the solution of the previous incubation. The instrument accommodates two thermoracks for the simultaneous processing of 96 slides.

The results described in this report have been generated by the group of Professor Gregor Eichele, Hanover. Examples presented are the expression of calbindin in the adult mouse brain and of neurotrophic tyrosine kinase type 2 receptor (Ntrk2) in a 14.5-day-old mouse embryo. More results of the groups large-scale mouse gene expression project can be viewed at www.genepaint. org. The nonradioactive automated in situ hybridization method and its application have been described previously [1, 2, 3].

Materials and Methods

 Preparation of DIG-labeled RNA probe

 The DNA template corresponding to the gene of interest is produced by polymerase chain reaction (PCR) using a gene-specific primer pair that also comprises the T3, T7, or SP6 promoter sequences.
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Mouse polyclonal antibody to NR2F2 - nuclear receptor subfamily 2, group F, member 2
The Discovery Series PDQuest 2-D analysis software is used for imaging, analyzing, and databasing two-dimensional electrophoretic gels. The 10-user network license entitles ten users to access data f
Mouse monoclonal antibody raised against a partial recombinant NEK2. NCBI Entrez Gene ID = NEK2
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