Immunocytochemistry of the amphibian embryo--from overview to ultrastructure.
Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.
Kurth
Thomas
T
Max-Planck-Institut für Entwicklungsbiologie, Abteilung Zellbiologie, Tübingen, Germany. Thomas.Kurth2@mailbox.tu-dresden.de
D000818:Animals; D004625:Embryo, Nonmammalian / Q000502:physiology / Q000648:ultrastructure*; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D009195:Peroxidase / Q000378:metabolism; D010969:Plastics / Q000737:chemistry; D013997:Time Factors; D014982:Xenopus laevis / Q000196:embryology*
D010969:Plastics; D009195:Peroxidase