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Embryonic lung explant culture

VH Vera Hutchison
AL Anne Lynch
AG Andrés Mauricio Gutierrez-Gamez
JC Jichao Chen
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Time-mated females were harvested at E14.5 and lungs with trachea and glottis were removed from embryos. The Cre-recombined ROSA-Kaleidoscope plasmid and Fast Green FCF dye (MKCD1540; Sigma-Aldrich) were mouth-pipetted through the glottis until embryonic lumens were visibly filled. Lungs were electroporated in BioRad Xcell electroporation cuvettes (165-2088; BioRad) at 35 V for three rounds of 25 ms with 1-s intervals. Lungs were then placed on Nuclepore Track-Etch Membrane filters (10417101; Whatman) and floated on RPMI-1640 media with 10% FBS 1% PenStrep for 24 h before fixation in 0.5% PFA for 4 h. After PBS wash, lungs were embedded in optimal cutting temperature compound (OCT; 4583; Tissue-Tek), cryosectioned, and imaged on Deltavision Deconvolution scope with 60× oil objective (Plan-Apo NA 1.42) running SoftWoRx.

Copyright and License information:  ©2023 Hutchison et al.
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