A1r si hd confocal microscope

Quantitative assessment of gap junction activity was performed using FRAP as previously described (Kuzma-Kuzniarska et al., 2016 ). Briefly, epithelial tissues were treated with calcein-AM (Thermo Fisher Scientific) for approximately 30 min, then excess dye was washed away using fresh culture medium. FRAP was performed on a Nikon A1R-Si HD confocal microscope. Whole cells were photobleached, then monitored for fluorescence recovery over a 4-min period. Recovery traces were plotted using minmax normalization. Cells in the center of the tissue (“inner”) were compared with cells located one cell layer in from the periphery of the tissue (“outer”), to control for number of neighbors.

Fluorescent images of embryonic lungs were captured using a spinning disk confocal (X-Light V2tp, Crest Optics) fitted to an inverted microscope (Nikon Eclipse Ti) with either a 10× air, 20× air, or 40× oil objective. Fluorescent images of adult lungs were captured using a Nikon A1R-Si HD confocal microscope with a digital micromirror device and 10× air objective.