Dm6000b inverted microscope

Brightfield images of lungs from in vivo and in vitro experiments were captured either on a Leica MZ 125 stereoscopic dissecting microscope using a Spot Insight 2.0 Mp Color Mosaic camera and Spot 4.5.9 imaging software, or were obtained from live imaging experiments using a Leica DM6000B inverted microscope, DFC 305FX camera, and Leica Application Suite Advanced Fluorescence imaging software.

Brightfield images of lungs from in vivo and in vitro experiments were captured either on a Leica MZ 125 stereoscopic dissecting microscope using a Spot Insight 2.0 Mp Color Mosaic camera and Spot 4.5.9 imaging software, or were obtained from live imaging experiments using a Leica DM6000B inverted microscope, DFC 305FX camera, and Leica Application Suite Advanced Fluorescence imaging software. Fluorescence intensities were quantified using FIJI software (version 2.0.0-rc-68/1.52 g).
Branching was quantified by counting and averaging the distal tips of the left lobe of control and experimental samples at 0 and 24 h. These groups were analyzed using a two-factorial quasi-Poisson model, with the dispersion parameter estimated as 0.317. Differences were considered statistically significant at p < 0.05.

Cultured cells were washed with PBS, fixed in cold 3.7% PFA for 15 min and re-washed with DPBS prior to staining. PBS-T (0.3% Triton-X-100 in PBS) was used to permeabilize cells for 10 min, followed by a 30-min block with 2% bovine serum albumin and 5% donkey serum (Gentaur). Cells were incubated overnight with primary antibodies in PBS-T with 5% donkey serum at 4°C. The subsequent day, cells were washed with PBS-T and incubated in the dark for 2 h with secondary antibodies (AlexaFluor anti-donkey 488, 555, 647; Life Technologies) diluted in PBS-T at room temperature. DAPI (Sigma), diluted 1:3000 in PBS, was used for nuclear staining. Stained cells were imaged using a Leica DM6000B inverted microscope, an average of 10 random fields of view for each staining combination at ×20 magnification. Cells were counted either manually (cytoplasmic markers) or using automated Cell Profiler (nuclear markers). N-cadherin was used for apical localization of the neural rosettes, with rosette size measured manually using FIJI (ImageJ). Immunocytochemistry quantification was collected from at least three biological replicates from at least three independent experiments for each marker. Antibodies are listed in the Supplementary material and Supplementary Methods Table 1.