PCLSs provided a 3D cell culture model to image vascular remodeling within the lung microenvironment and were adapted from previously described protocols (22 (link), 58 (link)). For mouse PLCSs, lungs were inflated in situ with 0.4 ml of 2% low-melting agarose (Thermo Fisher Scientific) in PBS. After inflation, lungs were carefully dissected out and fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) overnight at 4°C. Human lung tissue was collected from anonymized donors at Hammersmith and Royal Brompton Hospitals NHS Trusts and immediately fixed in 4% PFA overnight at 4°C. Following fixation, 100–200 μm transverse sections were prepared using a Compresstome VF-300 Vibrating Microtome (Precisionary Instruments).
PCLS were permeabilized in PBS complemented with 0.5% Triton (MilliporeSigma) for 1 hour at room temperature, then blocked in animal-free blocker (2BScientific Ltd.) for 1 hour. Slices were incubated with indicated primary antibodies overnight at 4°C in 25% animal-free blocker in PBS, and where required, PCLS were incubated with secondary antibodies for 5 hours at room temperature in 25% animal-free blocker in PBS. Lung slices were mounted on microscopic slides (Thermo Fisher), immersed in ProLong Diamond (Thermo Fisher), and kept at 4°C until image acquisition.
PCLS were permeabilized in PBS complemented with 0.5% Triton (MilliporeSigma) for 1 hour at room temperature, then blocked in animal-free blocker (2BScientific Ltd.) for 1 hour. Slices were incubated with indicated primary antibodies overnight at 4°C in 25% animal-free blocker in PBS, and where required, PCLS were incubated with secondary antibodies for 5 hours at room temperature in 25% animal-free blocker in PBS. Lung slices were mounted on microscopic slides (Thermo Fisher), immersed in ProLong Diamond (Thermo Fisher), and kept at 4°C until image acquisition.
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