The techniques for whole-mount in situ hybridization and antibody staining have been comprehensively reviewed elsewhere and, therefore, will not be discussed (Chapter 31, 12,13). However, some general pointers and pitfalls for the selection of markers can be outlined.
1. Care must be taken to use in situ probes or antibodies that are robust in their application. If this is the case, most protocols can be modified and considerably shortened without much loss of sensitivity.
2. Reagents can often be reused many times without loss of sensitivity of detection, and this sensitivity can actually increase with use. Since cost is a major factor in designing such a screen, with dioxygenin and antibodies being the major expense, this is an important consideration.
3. Since it is envisaged that a screen based on revealing gene patterns or specific proteins would utilize several markers at a time, care must be used to select individual markers that are within each other's range for sensitivity of detection. Also, each individual marker should mark distinct subpopulations of cells that do not obscure visualization via other markers.
4. For antibody stainings, fixation times for individual antigens may be incompatible. These parameters can often only be determined empirically.
5. Importantly, detection must be via simple inspection utilizing light microscopy, without the need for sophisticated mounting and visualization techniques, since this is preclusive to the ability to screen large numbers of individual broods.
6. Choose markers that reveal some aspect of developmental regulation that is not obvious on inspection. For example, somite formation is clearly visible within the transparent embryo, and markers that stain the whole of the developing somite may reveal less than simple inspection of the developing somite. However, a marker, such as krox 20, that stains specific rhombomeres in the hindbrain of the developing embryo that are difficult to visualize under the dissecting microscope may be more useful in a marker-based screen.
A large number of genes and proteins have been identified in zebrafish that fit these basic criteria. Examples of markers that can be utilized in such a screen are given in Table 1. This is by no means an exhaustive list, with the number of possible markers growing fast, and it is meant only as a guide to demonstrate the possibilities of this approach. Although the number of mutations that directly affect the expression pattern or distribution of gene transcript and proteins of interest will only be a small subset of the mutations revealed, this type of approach immediately allows the assessment of a particular mutation and its possible involvement in a process of interest.
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