3.4.1. Fixation of the Experimental Embryos
Host embryos can be fixed from several hours after the operation to several days after hatching according to the experimental design (see Note 20). Carnoy fluid is one of the best fixatives utilizable at once for Feulgen and Rossenbeck staining (13), immunohistochemistry, and in situ hybridization on paraffin sections. Fixation with 4% paraformadehyde is used for whole-mount immuno-histochemistry and in situ hybridization, and all treatments on cryostat sections.
3.4.2. Feulgen and Rossenbeck Staining
The Feulgen and Rossenbeck nucleal reaction (2,13) (see Note 21) is applied on 5-pm serial sections.
1. Two antibodies recognize virtually all cell types in the quail and no one in the chick: the polyclonal antibody raised by Lance-Jones and Lagenaur (33) and the monoclonal antibody (MAb) QCPN prepared by B. M. Carlson and J. A. Carlson, which is available at the Developmental Studies Hybridoma Bank (Department of Biology, University of Iowa, 436BB, Iowa City, IA 52242). The use of QCPN is easy and can be combined with other antibodies like HNK1 (34), which recognizes neural crest cells or 13F4 (35), which marks muscle cells and their precursors.
2. Other MAbs are species- and cell type-specific: MB1 and QH1 (36,37), which recognize a glycosylated epitope carried by surface proteins expressed in quail leukocytes and endothelial cells at the exclusion of any cell type of the chick.
3. Neural chimeras can be analyzed with MAbs that recognize either neuronal cell bodies or neurites of quail or chick exclusively (38,39).
A growing number of species-specific nucleic probes can be used on sections or whole-mount preparations. As examples, the use of a chick probe of the homeobox gene goosecoid has demonstrated the induction of this gene in a chick in which goosecoid-producing tissues had been grafted (40). The quail specific SMP (Schwann cell myelin protein) probe (41) allows quail oligodendrocytes in chimeric spinal cord to be distinguished (15) (Fig. 1C). Chick Wntl and quail Wntl probes have been combined to demonstrate the induction of Wntl in quail-chick chimeras (42).
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