Practical Considerations

It is essential to examine wild-type and mutant ovaries in parallel and under optimal conditions: in well-fed females mated with wild-type or sterile males (for unfertilized eggs). When a new mutation is suspected to be involved in oocyte/egg development, a fertility test should be performed to assess egg morphology and production rate in comparison with wild type. Where mutant females do not lay eggs or the morphology and/or production rate of eggs are apparently altered, then ovarian morphology should be analyzed. For this purpose, ovaries should be dissected from both wild-type and mutant females of the same age and analyzed under a dissecting microscope. Based on morphology, mutations affecting oocyte/egg development can be categorized as rudimentary, tumorous, degenerating, small egg, cup or open chorion, dumpless, dorsalized, ventralized, fused filament, thin chorion, collapsed egg and heldegg mutations (3). After this examination, usually enough information is gathered to decide which developmental stage(s) and/or process(es) should be analyzed by immunostaining.

The immunostaining methods for oogenesis are based on noncoagulant fixatives like methanol, acetone, formaldehyde, and paraformaldehyde, which rapidly penetrate and convert the cytoplasm into an insoluble gel and render the chromatin resistant to extraction by phosphate-buffered saline (PBS) and Triton X-100 or Tween-20. In general, the immunostaining methods presented in this chapter perform well in various laboratory conditions and with many of the antibodies listed in Appendix B. When new antibodies are tested, it is important to check several fixation conditions and antibody dilutions. By combining these two factors, one should be able to determine the optimal fixation parameters and antibody dilution for a novel antibody.

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