A1rsi inverted laser confocal microscope

Lungs from E16.5 and E18.5 embryos were fixed in 4% paraformaldehyde, embedded in paraffin or OCT compound and sectioned at 5 or 7μm respectively. Histological staining and immunohistochemistry were performed on paraffin sections according to standard protocols. TUNEL staining was performed on paraffin sections using ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Sigma Aldrich). Bright-field images were obtained using a Zeiss Axio ImagerA2 microscope equipped with AxioVision Software. Immunofluorescence staining was performed on E16.5 lung cryosections and fluorescence images were obtained using a Nikon A1Rsi inverted laser confocal microscope.

Cell proliferation in E16.5 lung cryosections was characterized by BrdU and phospho histone H3 immunofluorescence and quantified in Nikon Elements General Analysis 4.50. For BrdU labeling, E16.5 pregnant mice were injected intraperitoneally with BrdU at 50 mg/kg of body weight and the animals were sacrificed 2 hours after the injection. For quantification of immunofluorescence staining, tile scans of the whole lung were taken on a Nikon A1Rsi inverted laser confocal microscope; approximately 10, 40× random regions were selected. In each image, an average of 1,000 cells was counted for analysis. A total of 10,000–15,000 cells were counted per mouse. A 2-tailed Student’s t-test was used to determine statistical differences between genotypes. Cell death in E16.5 paraffin sections were characterized by TUNEL assay and Caspase-3 immunohistochemistry and quantified in Nikon Elements General Analysis RGB 4.50. Widefield tile scans were taken of the whole lung on a Nikon Eclipse Ni-E upright microscope; 10–15, 20X random alveolar regions were selected. In each image, an average of 2,000 cells was counted for analysis. A total of 20,000–30,000 cells were counted per mouse. A 2-tailed Student’s t-test was used to determine statistical differences between genotypes.