After the desired period of incubation, fix the embryo by flooding with 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). In the case of methanol or Zenker's, which are "rapid" fixatives, it is advantageous first to submerge the cultured embryo in saline, then to detach it from the vitelline membrane and to transfer it to a clean dish with saline prior to fixation. Otherwise the embryo will adhere permanently to the membrane and the fixative will denature the albumen, generating threads of protein that will tend to stick to the embryo. In the case of formaldehyde, this can be poured directly onto the embryo provided that the embryo is then detached from the membrane within a few minutes.
Whatever the fixative and subsequent method of processing chosen, it is advantageous to ensure that at the time of fixation the embryo is as flat as possible. If it has been detached from the membrane prior to fixation, place it in a small drop of saline on a plastic surface, then suck off most of the saline with a fine Pasteur pipet so that the embryo flattens on the plastic, and then place the first drop of fixative directly onto the surface of the embryo, taking care not to break it. After this it is safe simply to submerge the embryo in fixative, perhaps transferring it to a glass vial. Embryos fixed in this way usually remain flat through all subsequent manipulations.
Depending on the purpose of the experiment to be performed, embryos operated and cultured as described can be subjected to histochemistry, immunological procedures (as whole mounts), or whole-mount in situ hybridization. In many cases, it is possible to combine two or more of these methods. For example, it is possible to fix the embryos in formaldehyde, process them as whole mounts for in situ hybridization, postfix in formaldehyde, and then perform whole-mount immuno-peroxidase histochemistry with QCPN antibody to detect the quail cells. After this, they can be embedded in wax and sectioned. Methods for this have been published in this volume elsewhere in some detail (see Chapter 22 and ref. 17).
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