Metamorph 7 52

After decellularization, thin cut lung slices were incubated in 1mg/ml collagenase Type IV (Worthington, Lakewood, NJ, USA) for 7 days at 37°C on a vibrating plate. Collagenase media was changed every 30 minutes for the first 2 hours, then every hour for the next 2 hours, then every 24 hours thereafter. The colllagenase treated lung was stained with 5-fold excess of rabbit polyclonal anti-elastin antibody (Biorbyt, Cambridge, UK). Both anti-elastin and control conditions were treated with a fluoresceinated goat polyclonal anti-rabbit antibody (Pierce Antibody Products, Rockford, IL, USA). Serial stained sections were examined by flourescence grid confocal microscopy (Lee et al., 2009 (link)), processed in parallel and analyzed using MetaMorph 7,52 (Molecular Devices, Downingtown, PA) intensity applications as previously described (Gibney et al., 2012 (link)).

Analysis of still and video images was performed with MetaMorph 7.52 (Molecular Devices, Downingtown,PA) as previously described (Lee et al., 2011 (link); Lee et al., 2010 (link)).The grayscale images were processed with standard MetaMorph filters. After routine thresholding, the image sequences were measured using MetaMorph's integrated morphometry and caliper applications. Morphometric measurements such as EDD 10-14 embryo and CAM surface area, and CAM perimeter were calculated using serial, distance-calibrated images of the ex ovo CAM obtained orthogonal to the CAM surface.
Vessel interbranch distance and branching angle were similarly measured with digital morphometry applications and fluorescent micrographs. Interbranch distance was defined as the length of the vessel segment between two vessel branch points. Vessel branch angles were measured as the angle between two branching vessel segments Capillary plexus fractional area was calculated as the proportion of pixels representing capillary plexus vasculature in binarized, high resolution SEM images. Pillar density in the SEM images was determined after counting the number of intravascular pillars (intravascular channel or “hole”-like structures less than 2.0 μm) present in 100 μm² grid area. The pillar number per 100 μm² is reported after adjusting for capillary plexus density.