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

Abstract

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 Ca 2+ 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.

Mfge8 attenuates human gastric antrum smooth muscle contractions

“…For both immunofluorescence and isPLA the gastric antrum smooth muscle strips were fixed with 4% paraformaldeyde in PBS, and then cryo-protected with PBS/30% sucrose at 4 °C, embedded in OCT, and frozen at − 80 °C (Xie and Perrino 2019). The blocks were cut using a microtome into 10 µm sections and placed onto Vectabond (SP-1800) coated glass slides (Fisherbrand Superfrost Plus Microscope Slides, 12-550-15).…”

“…sigma aldri ch. com) (Xie and Perrino 2019). The muscle sections were incubated with each primary antibody (1:400 dilution) sequentially for 1 h at room temperature.…”

“…olymp us-lifes cience. com) (Xie and Perrino 2019). Confocal micrographs are digital composites of the Z-series of scans (1 µm optical sections of 10 µm thick sections).…”

“…The PLA figures were created from the digitized data using Adobe Photoshop Version 12.0.3. Fiji software was used to count PLA spots (Xie and Perrino 2019). Graphs were generated using GraphPad/Prism.…”

Increased talin–vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections

“…1e). Xie and Perrino [37] reported improper antibody dilution and insufficient washing between steps during staining can lead to false positives. In addition to routine standard rinse procedures between incubations (see "Materials and methods"), we performed antibody titration assay for optimal working concentrations ( Fig.…”