Paraformaldehyde solution

The lung tissue sections were prepared for immunofluorescent staining by performing deparaffinization, rehydration, and antigen unmasking as described by Crosby et al.⁵⁰ .
Normal control and COPD cells at weeks 1 to 3 ALI were fixed using 4% Paraformaldehyde solution in PBS (ThermoFisher Scientific, MA, USA). Sucrose infiltration steps were performed on normal control and COPD cells¹⁵ , followed by embedding in a biopsy-size cryomold using Optimal Cutting Temperature (O.C.T) compound (Tissue-Tek, CA, USA) and cutting them to 10 µm sections using Leica Cryostat CM3050 S (Leica Biosystems Inc., IL, USA). The sections were attached to Superfrost Plus microscopic slides (Fisher Scientific, PA, USA) and dried at room temperature overnight.

After live imaging, the chips were fixed using a paraformaldehyde solution 4% (Thermo Fisher Scientific). Thirty minutes later, all channels were flushed with PBS. Using a vibratome (Thermo Fisher Scientific, Microm HM 650 V), the chip was then cut into crosswise slices of roughly 200 mm, which were, in turn, permeabilized for an hour at room temperature (RT) using a buffer [PBS/0.2% Triton X-100/0.5% bovine serum albumin (BSA)].
The transversal sections in fig. S1 were first permeabilized with a 1% solution of saponin (Sigma-Aldrich) in 1× PBS for 1 hour at RT. They were then washed three times with PBS and incubated with a 2% solution of BSA (Sigma-Aldrich) in 1× PBS (2 hours, RT). After washing, the slices were incubated with SiR-Actin diluted 1/1000 in a 1× PBS solution of 2% BSA and 1% saponin (1 hour, RT). The resulting samples were washed an additional three times and conserved in PBS at 4°C. We remark that SiR-Actin binds F-actin, which is present inside the brush border of the microvilli. All prepared sections were imaged using the same confocal microscope described above, alternating between a 20× and a 40× (CFI Apochromat Lambda S 40XC water immersion with an NA of 1.25) objective.

We allowed 5×10⁵ SaOS-2 and MG-63 cells to grow in six-well plates in a monolayer for 24 hours. Cells were washed with RPMI-1640 medium and incubated with L-DOX (10 μg/ml) for transfection for 3 hours at 37˚C. The cells were rinsed three times with PBS and fixed with a 4% paraformaldehyde solution (Thermo Scientific, USA). For nuclear counterstaining, we used DAPI (0.125 µg/mL) for 15 minutes. Cellular uptake of DOX was evaluated with a fluorescence microscope (BX61, Olympus, Japan).