Protocol for in situ Proximity Ligation Assay (PLA) and Microscopy Analysis of Epidermal Growth Factor Receptor...

Oncogenic drivers play central roles in tumorigenesis as well as in tumor cell survival and proliferation. Mutations of the epidermal growth factor receptor gene (EGFR) that result in constitutive activation of the receptor tyrosine kinase have been identified as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). PCR-based assays are usually adopted for the detection of EGFR mutations, but no methods to detect EGFR activation that are not based on mutation identification have been established in the clinical setting. We describe a proximity ligation assay (PLA) used to visualize and quantitate EGFR homodimerization in NSCLC cell lines and tissue specimens. Rabbit monoclonal antibodies against EGFR were conjugated to PLUS or MINUS PLA oligonucleotide arms using Probemaker. Annealing of the PLUS and MINUS PLA probes occurred when two EGFR monomers were in close proximity, and repeat sequences in the annealed oligonucleotide complexes were amplified then recognized by a fluorescently-labeled oligonucleotide probe. PLA signals were detected and counted with a fluorescence microscope. We demonstrate the detection of EGFR homodimers by PLA analysis in a quantitative manner in both NSCLC cell lines and tissue samples obtained by transbronchial lung biopsy. PLA methods are a new tool for the detection and quantitation of protein-protein interactions such as homodimers, heterodimers, and fusion proteins.