Proximity ligation assay (PLA)

An in situ PLA is a modification of traditional immunoassays, which is capable of detecting protein-protein interactions with high specificity and sensitivity (29). Two antibodies are used in these assays, which are raised in different species against the target antigen. PLA probes facilitate the binding of the primary antibodies to the secondary antibody only when PLA probes are in close proximity. Each interaction between the two proteins results in bright fluorescent dots. We used a PLA to demonstrate protein-protein interaction suggestive of a complex formation between ADM2 and CRLR using a Duolink II fluorescence kit (Olink Biosciences) according to the manufacturer's instructions. Briefly, first-trimester HTR-8/SVneo cells sparsely grown on 16-well Lab-Tek chamber slides were treated with or without ADM2 (10⁻⁸ M) for 2 minutes, fixed using 4% paraformaldehyde, and incubated with primary antibodies (one monoclonal antibody and one polyclonal antibody) followed by incubation with PLA probes. A PLA on the villous tissue was performed in a similar way on frozen sections after fixation in paraformaldehyde and antigen retrieval with 1% sodium dodecyl sulfate (SDS). Images are observed under a fluorescence microscope (U-TV1 X; Olympus), and red spots were counted using Image-Pro Plus software (Media Cybernetics) in 10 randomly selected images per replicate. Appropriate negative controls included no primary antibody or a negative IgG isotype of primary antibody.