B

Fig. 1. Apoptosis in the rhombencephalic neural crest of the chick embryo as revealed by acridine orange staining (A,B) and TUNEL (C,D). The embryos shown in (A) and (C) are at stage 10, but those in (B) and (D) are older, stage 11 (see Note 1 and color plate 3 appearing after p. 368).

in this system is that by clearing these two territories (rhombomeres 3 and 5) of neural crest cells, the neural crests that emerge from the adjacent regions and that are spatially preprogrammed are kept separate from each other (3).

Acridine orange staining has the advantages that it is extremely rapid, cheap, and easy to perform. On the other hand, it has a number of disadvantages. Firstly, because it is used vitally, and since the dye is not fixable and consequently the stain is transient, the embryos cannot be processed for other detection procedures, such as immunohistochemistry. Acridine orange staining also has the drawback that the embryos must be processed swiftly and viewed almost immediately, at least within 1 h of staining, since the tissue dies and the intense staining of the apoptotic bodies is lost. It should also be pointed out that although the signal may be more intense under flourescein epifluoresecne, necrotic cells will also be picked up under this illumination. This method also has the disadvantage that since there is no permeabilization step, apoptotic cells that lie deep within tissues may not be detected. One way of improving this is to cut the embryo prior to staining so that there is greater access to the tissue of interest. For example, with the central nervous system, one can slit the neural tube dorsally.

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