In situ proximity ligation assay. (1) Recognition and binding of oligonucleotide labeled antibodies to interacting target proteins. (2) Formation of covalently joined circular oligonucleotide by ligase reaction: If the two probes are in close proximity, addition of two linear oligonucleotide leads to the formation of a covalently joined circular oligonucleotide molecule with the help of a ligase enzyme. (3) Amplification via Rolling circle mechanism: One of the probes linked to antibody act as a primer and addition of DNA polymerase yields long single stranded concatemeric DNA molecule composed of complements of the circular DNA strands formed in the ligase reaction. (4) Fluorophore labeling for detection: The amplified product is easily detected by addition of complementary oligonucleotides labeled with fluorophore.