Immunoelectron microscopy in embryos.
Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.
Sierralta
W D
WD
Max-Planck-Institut für experimentelle Endokrinologie, D-30603, Hannover, Germany. wsierra@uec.inta,uchile.cl
D000818:Animals; D019521:Body Patterning; D002642:Chick Embryo; D004625:Embryo, Nonmammalian / Q000648:ultrastructure*; D005121:Extremities / Q000196:embryology; D019204:GTP-Binding Proteins; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods; D008648:Mesoderm / Q000378:metabolism; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009363:Neoplasm Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface
D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011493:Protein Kinase C; D019204:GTP-Binding Proteins