Notes

1. Fig. 1C,D shows the results obtained using TUNEL, and streptavidin-horserad-ish peroxidase detection, on whole chick embryos during the period of hindbrain neural crest apoptosis. As was found with the acridine orange staining, during the early phase of crest production, there is staining over rhombomere 3 (Fig. 1C), and at a slightly later stage, this staining is more pronounced and extensive, and also apoptotic cells are also found over rhombomere 5 (Fig. 1D).

2. The disadvantages of using TUNEL are that it is relatively expensive and takes somewhat longer than the acridine orange staining, but it has many advantages that together make it the method of choice. One big advantage is that this method uses embryos that are fixed. This means that one can accumulate specimens at 4°C and that they can be processed at leisure. The fixation also allows one to permeabilize the embryos using detergent, Triton X-100, and therefore even apoptotic cells deep within the embryo can be detected. The fact that this method can use fluorescein or horseradish peroxidase detection also means that this technique can be coupled with other procedures, such as immunohistochemistry or axonal tracing, and at least when using horseradish peroxidase detection, the preparations are permanent. The horseradish peroxidase-reacted embryos can also be sectioned afterward to allow a more complete analysis of the distribution and the cell type of the apoptotic cells.

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