title: Applying the proximity ligation assay (PLA) to mouse preimplantation embryos for identifying protein-protein interactions in situ.
abstract: Analysis of protein-protein interactions in mouse preimplantation embryos is hindered by the low cell number of the embryo. Here we describe the use of the proximity ligation assay to overcome these limitations and outline how protein-protein interactions can be visualized in situ. The method is based on a normal immunofluorescence labeling protocol of preimplantation embryos. Events of binding of the two primary antibodies directed against two individual proteins close to each other are visualized. If the two primary antibodies and the corresponding oligo-linked secondary antibodies bind in close proximity a cascade of events is initiated. This includes oligo ligation, DNA amplification, and hybridization with a fluorescent probe that allows visualization of this close proximity. In contrast to normal immunofluorescence labeling, here detection of red fluorescent dots reflects protein-protein interaction.
authors.affiliation: Department of Molecular Embryology, Max-Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany.
authors.forename: Ivan
authors.initials: I
authors.lastname: Bedzhov
authors.forename: Marc P
authors.initials: MP
authors.lastname: Stemmler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001755:Blastocyst / Q000378:metabolism* / Q000648:ultrastructure; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D025941:Protein Interaction Mapping; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; C495267:CTNNB1 protein, mouse; D015820:Cadherins; D051176:beta Catenin; D017526:Receptor, IGF Type 1