Acridine Orange Staining

1. Dissect out the embryos into an appropriate physiological salt solution, such as PBS, for mouse embryos or Howard's Ringer for chick embryos, and process directly.

2. Incubate embryos at 37°C in PBS, or Ringer's, containing acridine orange at a concentration of 5 ^g/mL for 15 min.

3. Wash the embryos twice, with shaking, for 1 min each in PBS.

4. Mount the specimens under a glass coverslip in either PBS or Ringer's and analyze the specimens immediately under rhodamine epifluoresence.

5. Photograph the results.

Results obtained with acridine orange staining of whole chick embryos are shown in Fig. 1. The images shown here were taken using a Bio-Rad MRC 600 (Bio-Rad, Hemel Hempstead, UK) confocal microscope, and the rhodamine and phase images have been merged to give a clear picture of the relationship between areas of acridine orange staining and embryonic anatomy. During the early phase of neural crest production in the chick hindbrain, a focus of acridine orange staining cells is observed over rhombomere 3 (Fig. 1A), whereas at slightly later stages, the focus over this segment is enlarged, and there is also prominent staining over rhombomere 5 (Fig. 1B). The importance of apoptosis

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