Quantitative in situ proximity ligation assays examining protein interactions and phosphorylation during smooth...

Antibody-based in situ proximity ligation assays (isPLA) have the potential to study protein phosphorylation and protein interactions with spatial resolution in intact tissues. However, the application of isPLA at the tissue level is limited by a lack of appropriate positive and negative controls and the difficulty in accounting for changes in tissue shape. Here we demonstrate a set of experimental and computational approaches using gastric fundus smooth muscles to improve the validity of quantitative isPLA. Appropriate positive and negative biological controls and PLA technical controls were selected to ensure experimental rigor. To account for changes in morphology between relaxed and contracted smooth muscles, target PLA spots were normalized to smooth muscle myosin light chain 20 PLA spots or the cellular cross-sectional areas. We describe the computational steps necessary to filter out false-positive improperly sized spots and set the thresholds for counting true positive PLA spots to quantify the PLA signals. We tested our approach by examining protein phosphorylation and protein interactions in smooth muscle myofilament Ca²⁺ sensitization pathways from resting and contracted gastric fundus smooth muscles. In conclusion, our tissue-level isPLA method enables unbiased quantitation of protein phosphorylation and protein-protein interactions in intact smooth muscle tissues, suggesting the potential for quantitative isPLA applications in other types of intact tissues.