Fig. 5 Schematic representation of the limb fillet technique. An incision is made along the posterior aspect of the limb bud, and the limb epithelium is flattened. After removal of the mesoderm, the Ti1 pathway is exposed and can be visualized. See text for details.
G. Labeling and Injecting Til Neurons in Situ
1. Limb fillets are prepared as described in Section V,F.
2. To label Ti1 cell membranes with a lipophillic dye (e.g., Dil or DiO), Ti1 cell bodies are located with Nomarski optics and then a dye-coated solid microelectrode is gently pushed up against the cell membrane using a micromanipulator. The electrode is left in contact with the Ti1 cell membrane for 10 s to 1 min, depending on the rate of transfer of dye from the electrode to the cell membrane.
3. Lipophillic dye-coated electrodes can be prepared a variety of ways; however, we find the following method to be the most reliable. A small solid piece of Dil or DiO (Molecular Probes) is placed in a small (^700 ^l) Eppendorf tube and 300-400 ^l of distilled water is added. The tube is then sonicated for 3-4 min in a pen-cleaning sonicator until there is an even dispersal of the dye. While sonicating, 5-10 solid micropipettes are arranged with their tips converging at the same point. Approximately 10 ^l of the sonicated dye is then placed on the pipette tips, and water is allowed to evaporate (15 to 20 min), resulting in a fine coating of dye. The solution of dye can be maintained in a —20° C freezer between applications; however, we find sonicating before application to be beneficial. In addition, coated pipettes become increasingly more effective with the frequency of coating.
4. Microinjection of Ti1 neurons is performed with a picospritzer. For the injection of large proteins and plasmids, we find that beveled electrodes are beneficial. In addition, because the electrode has to penetrate through the Ti1 cell membrane and the overlying basal lamina, we find that beveled electrodes do not clog as easily during injection attempts. Typically we find that a continuous flow at low pressure and a slight tap of the microscope will result in reliably injected cells. A disadvantage to this approach is that it is difficult to determine the volume of injected material.
H. Ectopic Overexpression in the Developing Limb Bud
1. Dissect embryos for culturing using sterile conditions as described and prepare a glass bottom dish.
2. For intact limbs, embryos are placed ventral side down onto an attached coverslip in a glass bottom dish filled with grasshopper culture medium as described earlier. The limbs are splayed away from the body wall. For filleted limbs, prepare as described previously.
3. Transfer transfected cells into a freshly pulled mouth micropipette (or, for a limb fillet, clustered cells can be transferred directly to the culture dish with a 10-ul pipette tip). We use thin-walled, 1-mm-diameter pulled micropipettes placed in a micromanipulator. The very tip is broken off to allow for the gentle expulsion of cells.
4. For intact limbs, insert the tip through the dorsal closure into a limb in a medial-to-lateral orientation. Cells are released into the limb with gentle pressure. Pipette placement and delivery of cells are monitored with a dissecting microscope.
5. For limb fillets, cells are expelled on, or near, the filleted limbs, and the clumps of cells are maneuvered individually with hand-held solid needles and tampers. This allows for precise placement of the cells along the Ti1 pathway. Cell placements are monitored with a dissecting microscope.
I. Blocking Protein Function
1. In general, antibodies should be affinity or IgG purified, followed by dialysis against grasshopper culture medium (see the aforementioned recipe). If desired, Fab fragments of antibodies can be prepared. The concentration of antibody used depends on numerous factors, including affinity for antigen and ability to bind in vivo. Previously, the 1-5 yM range has been effective at blocking.
2. Embryos are dissected and added to culture medium containing various concentrations of antibodies. Preliminary experiments using a range of antibody concentrations are used to establish the effective concentrations. Control antibodies should be used at the same concentrations and ideally should consist of antibodies generated to different regions of the same protein.
1. Peptides are dissolved in sterile H2O or suitable solvent.
2. It is assumed that peptides compete with endogenous protein activity. Effective concentrations vary and are generally established empirically.
We would like to thank members of the O'Connor lab for the critical reading of the manuscript. Work described in this chapter is supported by grants from the Canadian Institutes of Health Research (MOP-13246), the Natural Sciences and Engineering Research Council (OGP0171387) and the National Neurotrauma/Rick Hansen Institute (#99019990). TOC is an EJLB research scholar.
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