Anti egfr mouse monoclonal antibody

Proximity Ligation Assay (PLA), also called in-cell co-IP assay, by Olink Bioscience (Sweden), was used to identify specific colocalization events of CXCR7 and EGFR in fixed cells or tissues. Prior to the PLA, cells were fixed in 4% paraformaldehyde at 4°C, 15 minutes and human breast tissues were de-waxed. Cells and tissue were incubated with anti-CXCR7 rabbit IgG (1:500, GeneTex) and anti-EGFR mouse monoclonal antibody (1:500, Sigma) or normal rabbit IgG. Following incubation with primary antibody, cells were incubated with corresponding secondary antibodies that were conjugated with oligonucleotides (PLA probe MINUS and PLA probe PLUS). Then, ligation solution was added, consisting of ligase and two oligonucleotides that hybridize to the two PLA probes and form a closed circle if the PLA probes are in close enough proximity. Strand extension by rolling circle amplification (RCA) by T4-ligase and PCR amplification of double hybridized DNA was performed as described by the supplier. Fluorescently labeled oligonucleotides were used detection of the RCA product. The resulting signal (a red fluorescent signal of Texas-Red fluorescent tagged amplified DNA) occurs wherever the two molecules are colocalized. Quantification of the confocal images was performed using the Duolink Image Tool software (Olink Bioscience).