Development of a latex-conjugated immunocytological marker for scanning electron microscopic analysis of quail-chick chimeras.

Quail-chick chimeras are created by transplanting pieces of quail tissue to chick. We have used antibodies combined with a cell surface marking technique [modified after Molday et al.('75)], to facilitate scanning electron microscopic (SEM) identification of quail cells in such chimeras. First, antibodies to quail RBCs were raised in rabbits by intravenous injection. Rabbit anti-quail RBC serum was precipitated by ammonium sulfate, purified by DEAE chromatography, and cross-absorbed with chicken RBCs. Second, sheep anti-rabbit IgG was purchased commercially and further purified by affinity chromatography. Third, 900-A-diameter latex beads were synthesized by aqueous emulsion copolymerization of methacrylates. Spheres were bound with diaminoheptane to create an extension arm which was further derivatized in a two-step glutaraldehyde procedure. Purified sheep IgG was bound to the aldehyde-activated spheres, with uncoupled sheep IgG removed by sucrose density centrifugation. To test the marker, rabbit anti-quail IgG was added to 1% diluted quail RBCs. After washing, sheep anti-rabbit IgG bound spheres were introduced. Washed cells were fixed in one-half strength Karnovsky's and processed for SEM. Quail RBCs were uniformly decorated with beads, containing 2,000 beads per cell. Similarly treated chick RBCs show no binding to beads. Likewise, quail RBCs not pre-treated with the rabbit IgG do not bind beads. Prefixed quail RBCs still bind latex-conjugated beads, although at somewhat reduced levels. When mixtures of quail and chick RBCs were processed for identification: (1) sphere labeling was an "all or none" phenomenon; (2) the proportion of bead-decorated cells observable in the SEM was the same as the proportion of quail RBCs provided in the initial mix; and (3) morphologically distinguishable embryonic chick RBCs did not label whereas under the same conditions, quail RBCs do. We further demonstrate that rabbit antibodies prepared by injection of stage 4 quail primitive streaks can be used to specifically label quail epiblast and mesoblast cells, providing markers for at least two germ layers. It is now possible to combine grafting techniques of known success, with SEM analysis of the chimera.