Our objective is to develop new in situ (hybridization and immunocytochemistry) assay technology to take full advantage of the new generation of reagents for detecting low abundance regulatory macromolecules in cells. In situ assays often defer to biochemical assays even though they are better suited, because they are less convenient and reliable. Standard in situ methods are capillarity-based open chamber methods characterized by handling and dehydration artifacts, poor temperature control and many manual steps. Automated units that are now available work on the same principle, and consequently do little more than compound the problems intrinsic to the capillarity design. The """"""""Probe-Clip"""""""" reaction unit deviates from existing models in six key aspects: 1) the reaction chamber is closed rather than open; 2) there are tow chambers rather than one; 3) """"""""in chamber"""""""" reactant mixing is by gravity rather than capillarity; 4) reactants are conveyed to the chamber in solid rather than liquid state; 5) washing is in a high ambient volume closed chamber rather than a fast-flowing open stream; and 6) temperature control is by conduction rather than by convection. These changes give numerous advantages related to speed, safety, versatility and sensitivity. In Phase I, we will build a six-place manually operated prototype for the research laboratory, to validate the design concept for an automated model to be developed in Phase II.
