Embryos, operated as described in Subheading 3., can be studied by a variety of methods, according to the question being addressed. Fix the embryo in methanol (for most immunological detection procedures), Zenker's fixative (for Feulgen-Rossenbeck staining) or in 4% formaldehyde in PBS (for in situ hybridization and most other procedures). Details on these procedures may be found elsewhere in this volume and in ref. 9.
For embryos operated in ovo, it is easiest to crack the egg into a large Petri dish first, cut the membranes around the embryo with scissors, and then lift a corner of these membranes with fine forceps (as described above in Subheading 2.2.1.). Then immediately transfer this to a small dish with saline to clean off any adhering yolk. Finally transfer it to a Sylgard dish (see Subheading 2.1.) containing CMF and pin the embryo so as to straighten out the head and trunk, but avoiding the region close to the operation. Then remove the CMF and replace it with the fixative of choice. In this way, the embryos will be perfectly straight which will simplify subsequent histological sectioning, and they will also be more photogenic if stained as whole mounts.
For embryos operated in New culture (3-5), the glass ring should first be flooded with CMF, and the edges of the area opaca then detached from the vitelline membrane. Then pick up the embryo with a wide-mouthed pipet or with fine forceps (from the membranes!) and transfer it to a Sylgard dish for pinning and fixing as described in Subheading 2.2.1.
Was this article helpful?