Separation of Ectoderm Mesoderm and Dissection of Apical Ridge

1. Sterile phosphate-buffered saline (PBS).

2. Sterile ice-cold tissue-culture medium containing serum (for example, Minimal Essential medium + Hank's salts buffered with HEPES + antibiotics + fetal calf serum).

3. Sterile ice-cold 2% trypsin. (Recently, we have been using crude type II from porcine pancreas made up in calcium-magnesium free saline; pH to 7.5) store in aliquots at -20°C.

4. Ice-cold slab (a freezer block covered with black plastic will do).

5. Sterile pipets for transferring tissue.

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