Buccal mucosa smears were immunostained with the following primary antibodies using established protocols: mouse monoclonal anti-plakoglobin (P8087, Sigma Aldrich), mouse monoclonal anti-plakophilin-1 (325700, Thermo Fisher Scientific), mouse monoclonal anti-desmoplakin (10R-D108AX, Fitzgerald), and mouse polyclonal anti-Cx43 (C8093, Sigma Aldrich). Slides were then incubated with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) for 2 h at room temperature and counterstained with DAPI to label nuclei. All immunostained preparations were imaged at x20 using a Nikon A1R confocal microscope.
We optimized our immunostaining protocol for each protein by first determining the lowest primary antibody concentration that produced strong signal in control buccal mucosa cells. These conditions were then applied throughout the study to determine whether cells from subjects from ACM families showed apparent reduction in junctional signal. Using this approach, we consistently observed either control levels of signal or a virtual absence of junctional signal for any given primary antibody. This ‘binary’ approach ensures reproducibility of our results and precludes the need for signal quantification. Each batch of ACM samples was immunostained alongside freshly-obtained smears from age-matched controls.
We optimized our immunostaining protocol for each protein by first determining the lowest primary antibody concentration that produced strong signal in control buccal mucosa cells. These conditions were then applied throughout the study to determine whether cells from subjects from ACM families showed apparent reduction in junctional signal. Using this approach, we consistently observed either control levels of signal or a virtual absence of junctional signal for any given primary antibody. This ‘binary’ approach ensures reproducibility of our results and precludes the need for signal quantification. Each batch of ACM samples was immunostained alongside freshly-obtained smears from age-matched controls.