Ethics approval
The Animal Care Committee at the University of Manitoba reviewed and approved all experimental procedures (19-010/1/2 (AC11436)). Rats were mated overnight and the next day was considered embryonic day 0 (E0).
Nitrofen rat model of CDH
We dissolved 100 mg of nitrofen in 1 ml of olive oil while protecting the nitrofen from light. The next day, the dissolved nitrofen was used to induce CDH and pulmonary hypoplasia by delivering it to randomly selected pregnant rats by oral gavage on E9. Dams were euthanized by CO2 at E18, and fetuses were removed. These samples came from two different litters for each experiment.
Ex Vivo Lung Culture and TO surgical technique
E18 embryos were obtained similar to what has been previously described [10] to mimic a similar time point, canicular – saccular stage of lung development, to FETO in human CDH (week 27-29) [3]. Lungs were excised one-by-one and placed in room temperature Advanced DMEM/F12 (Cat. No. 12634010, Thermo Fisher Scientific) with 8mM L-glutamine and 100mg/mL primocin (Cat. No. ant-pm-1, InvivoGen). After dissecting the lungs, the trachea was tied with a 7-0 polypropylene suture (Cat. No. 18030-70, Fine Science Tools Inc.) for TO (Figure 1). Then, the explants were incubated on 12 mm Transwell® with 3.0 µm Pore Polyester Membrane Insert (Cat. No. 3462, Corning Incorporated) within DMEM/F12, 8mM L-glutamine, 100 mg/mL primocin, 1.0% FBS. The explants were cultured for 1–3 days.
Tissue processing
Lungs were formalin-fixed (3.7% formaldehyde) (Cat. No. 23–245-684, Thermo Fisher Scientific) at 4℃ overnight, then dehydrated in an ethanol gradient, cleared in xylene, and embedded in paraffin.
Figure 1
Immunostaining
Lung tissue sections were heated to 58℃ for 30 min and then deparaffinized using three xylene washes and rehydrated in a descending ethanol gradient before antigen unmasking in a pH 6 citrate-based solution (Cat. No. H-3300, Vector Laboratories) for 30mins in a boiling water bath. Sections were blocked and incubated with antibodies overnight at 4℃ in blocking buffer containing 3% FBS and 2% BSA in PBS with 0.1% Tween-20 (Cat. No. BP337-500, Thermo Fisher Scientific). The next day, the sections were incubated with Alexa Fluor secondary antibodies (Thermo Fisher Scientific) at room temperature for 3 hours. Nuclei were stained with DAPI and autofluorescence was reduced with 0.1% Sudan Black B (Cat. No. 199664-25 G, Sigma Aldrich) in 70% ethanol twice for 15 min each at room temperature. Sections were mounted in Fluoromount G (Cat. No. 0100-01, SouthernBiotech). Antibodies are shown in Table 1.
Image Quantifications
ImageJ was used for all image quantifications [11]. For the %Ki-67/DAPI+ and and %Active caspase-3/DAPI+, nuclei were counted with the Analyze Particles function. Prosurfactant Protein C (proSP-C) protein abundance was defined as the mean gray value of the immunostained images.
Table 1
Statistical Analyses
Statistical analyses were performed with GraphPad Prism 9 (GraphPad Software). Continuous data were analyzed using an unpaired t test. A p value of less than 0.05 was considered statistically significant.