Ab40879

At the end of the culture period, the bioreactor was disassembled and tissue was sectioned and fixed in 10% natural buffered formalin for 4 h and placed in 70% ethanol overnight, followed by embedding and sectioning into 5 μm slices. Sections were stained with hematoxylin and eosin (H&E) to examine global tissue architecture and cellularity.
For immunofluorescent analysis, slides were deparaffinized using xylene and rehydrated in water. Antigen retrieval was performed in 1 mM EDTA, 10 mM Tris, and 0.05% Tween-20 buffer at pH 9 for 20 min at 75°C and allowed to cool to room temperature for 20 min. After blocking sections with PBS containing 10% FBS and 0.2% Triton X-100 for 45 min, primary antibodies were used against CCSP (07-623, 1:100; Millipore), Pro-SPC (ab40879; Abcam), AQP-5 (AB3559-50UL, 1:500; Millipore), PCNA (ab29, 1:500; Abcam), and PECAM-1 (ab28364, 1:100; Abcam) for 2 h at room temperature. After washing slides with PBS, corresponding secondary antibodies (Alexa Fluor 555) were used at 1:500 dilution for 45 min. Mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) was applied to slides to visualize nuclei, and coverslips were placed to seal slides. The slides were visualized using a Leica DMI6000 B fluorescence microscope.

Freshly isolated ATs (NS) or passaged ATs (DS) were grown overnight in 35-mm cell-culture microdishes (Ibidi GmbH, Germany). The confluent layer of cells was subsequently fixed with 2% paraformaldehyde (Thermo Fisher Scientific) in phosphate-buffered saline (PBS, Gibco) at room temperature for 30 min, washed with PBS, and incubated with a blocking solution of 2% bovine serum albumin (BSA) in PBS for 1 hr at room temperature. The blocking solution was removed, and the cells were incubated with the primary antibody (1:100 dilution in 2% BSA solution in PBS) overnight at 4°C. Antibodies used were anti-Podoplanin Monoclonal Antibody (eBio8.1.1 (8.1.1)), Alexa Fluor 488, eBioscience (ThermoFisher Scientific), and anti-pro-SPC antibody (ab40879, Abcam). The cell-culture microdishes were washed 3x in PBS, incubated with a fluorescent secondary antibody (Donkey anti-rabbit Alexa Fluor 568 (A10042 Thermo Fisher)) in a solution of 2% BSA in PBS for 1 hr at room temperature, then thoroughly washed with PBS and incubated with Hoechst 33342 nuclear staining dye (1:1000 dilution, Thermo Fisher Scientific) for 15–20 min for nuclear staining. Confocal images were obtained on a Leica SP8 microscope in the inverted optical configuration at the EPFL BIOP core facility.

Primary antibodies for immunoblotting and immunofluorescence were as follows: anti proSP-C, ab40879 (Abcam, UK; WB 1:1000, IF 1:200); anti Collagen1, 600–401-103 (Rockland, USA; WB 1:1000, IF 1:200)); anti fibronectin ((H-300) (sc-9068, SantaCruz, Heidelberg, Germany; IF 1:200); anti E-cad (610181, BD, Franklin Lakes, NJ, USA, IF 1:200); anti aSMA (ab5694, Abcam, UK, IF 1:100); anti β-actin, A3854 (Sigma Aldrich, UK; WB 1:25000). Alexafluor conjugated (488, 555 or 647) anti-mouse and anti-rabbit secondary antibodies (Thermo Fisher Scientific, Germany) were used for IF.

Three 8-week-old miR-200b^−/− or miR-200b^+/+ lungs were inflated intratracheally with 1 ml of 10% formalin via the cannula by gentle infusion. All tissues were fixed in 10% formalin for overnight. Lungs were embedded in paraffin after dehydration in a graded ethanol series followed by xylene. Immunofluorescence was performed on 5 μm sections using vimentin (Abcam; ab92547; 1:800), pro-Surfactant Protein-C (Abcam; ab40879; 1:800), pro + mature Surfactant Protein-B (Abcam; ab40876, 1:50) and twist (Abcam; ab50887; 1:200) antibodies as previously described⁴⁵ .

The CXCR4-overexpressing HUMSCs or control HUMSCs, grown on glass coverslips, and frozen mouse lung tissues, sectioned at 4 μm and placed onto glass slides, were incubated overnight with rabbit polyclonal anti-CXCR4 antibody (1 : 200, Boster, China, BA0761), or rabbit polyclonal anti-pro-SP-C antibody (1 : 250, Abcam, USA, ab40879), respectively, in darkness at 4°C, followed by incubations with Dylight 549 (1 : 100, Abbkine, USA, A23320) for 1 h at 37°C. Then the total cell nuclei of the HUMSCs and the lung tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The fluorescence images of EGFP, CXCR4, and pro-SP-C were detected using a confocal laser scanning microscope (Leica, Germany, TCS SP8).