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.