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A major advantage of immunogold cytochemistry is its amenability to quantitative analysis. As demonstrated above, this technique may be put to advantage to monitor receptor trafficking events following ligand-induced endocytosis with a spatial resolution impossible with any other method. The distribution of immunolabeled receptors may be assessed in a number of ways. Gold particles may be classified according to their association with specific subcellular structures and the number of labeled structures expressed as a proportion of total Figure 4.
Cells can be maintained in culture more than three weeks after plating. For our work it would be convenient to have a simple protocol that allows: (i) to transfect embryonic hippocampal neurons at different developmental stages including fully mature neurons; (ii) to transfect several cDNAs at the same time in parallel experiments instead of producing a recombinant virus for each construct; and (iii) to cotransfect two plasmids into the same neurons. Thus, we adapted a CP protocol for efficient transfection of hippocampal neurons (5).
7. Rinse 3 × 5 minutes with PBS. 8. Incubate with the FITC-tagged secondary antibody diluted 1:100 to 1:500 in PBS for 30 minutes at room temperature. 9. Rinse 3 × 5 minutes in PBS. 10. Mount the coverslips, cell-side down on glass slides with Aquamount and examine by confocal microscopy. FITC signal is imaged by exciting samples with 488 nm and Bodipy red signal by exciting samples with 568 nm. Protocol for Monitoring the Effect of Ligand Exposure on the Subcellular Distribution of Neuropeptide Receptors Principles of Technique The present technique applies standard pre-embedding immunogold procedures, as originally developed in V.