The core provides equipment and services to determine gene expression patterns in mouse tissues. Moreover, the core will collect (or assist in collecting) specimens (normal, mutant and pathologic samples), prepare frozen sections, carry out In Situ Hybridization (ISH) and document expression patterns by microscopy. In addition, and upon request, the core will produce templates and riboprobes utilized for the in situ hybridization experiment.

Because the high-throughput technology on which this core facility is based is one of a kind, we shall briefly describe those features that distinguish it from the standard ISH process.

High-Throughput In Situ Hybridization
All steps required for the ISH proper are carried out in an automated fashion. Cryostat sections are placed on a standard microscope slide that is subsequently incorporated into a flow-through chamber (Figure 1A). The chamber is placed into a temperature-controlled rack and solutions required for pre-hybridization, hybridization, and signal detection reactions are added to the flow-through chamber with an automated solvent delivery system (Figure 1B). Because of such automation, up to 192 slides can be processed in parallel allowing a daily throughput of hundreds of sections. Although this equipment can be used for hybridization with radioactive probes its power comes to the fore when hybridization is carried out with epitope-tagged riboprobes (e.g. digoxygenin) that are detected with a biotin-tyramine-based amplification step (e.g. Kerstens et al., 1995). This detection method exhibits sensitivity comparable to that obtained with radioactive riboprobes but has the benefit of providing cellular resolution and increased speed (Figure 1D, E and G).

Figure 1. Equipment developed for automated in situ hybridization and illustration of typical results.
A.		Flow-through hybridization chamber (200 µl volume) composed of a microscope slide loaded with tissue sections, a pair of two 80-µm thick spacers, and a 5-mm thick glass plate into which a depression has been cut. Slide, spacers and glass plate are fastened into a metal frame with a bracket. Depression and slide form a well into which solutions are delivered by a pipetting robot. Solutions initially flow through the chamber but once the well is drained, solutions are retained in the narrow slit of the chamber as a result of capillary forces.
B.		Hybridization and detection chemistry are performed on a Tecan Genesis pipetting robot platform equipped with racks for the hybridization chambers (1), reagent containers (2), and a system for controlling the temperature of the racks (3). Solutions are added with a set of 8 parallel pipettes (4).
C.		A Leica microscope equipped with a motorized stage and a CCD camera.
D.		Sagittal brain section of an adult mouse showing the expression of the tyrosine hydroxylase gene which is expressed at numerous sites including olfactory bulb (OB), striatum (ST), cortex (CX), substantia nigra (SN), and cerebellum (CB). To visualize expression, digoxygenin (DIG)-tagged riboprobes are first detected with a antiDIG antibody to which peroxidase is coupled. This enzyme is used to activate a tyramine-biotin conjugate which thereby gets covalently attached to proteins in the vicinity of the antiDIG antibody. Subsequently, biotin is detected with a steptavidine-alkaline phosphatase-based color reaction. The grid defines 32 images individually captured and then assembled into the composite. Each image was collected at 50 fold magnification and has a resolution of 3.3 µm/pixel.
E.		Blow-up of area boxed in D; this image has the same resolution as that in D.
F.		Expression of secreted frizzled related protein 2 (sFRP2) in the midbrain (MB) detected with a radioactive probe.
G.		Expression of sFRP2 in the midbrain (MB) detected with a digoxygenin-tagged probe. Note the size of silver grain clusters on top of cells in F is broader than the size of a cell as defined by the color precipitate in G.

© 2003 Baylor College of Medicine - Mental Retardation and Developmental Disabilities Research Center
Phone: (713) 798-7353    Fax: (713) 798-8704    Houston, Texas 77030    E-mail: mrddrc@bcm.tmc.edu
Modified February 2008