Dm2500 fluorescence microscope

In accordance with a previously published protocol.^{17 (link)}
Samples were washed with 1% Tris-buffered saline (TBS) and fixed using methanol–acetone (1:1) (Sigma-Aldrich, UK). Samples were washed and subsequently immersed in 500 μl blocking solution [5% goat serum (Gibco, UK) and 0.2% triton-X (Sigma-Aldrich, UK) in TBS]. After the blocking step, each well was incubated with rhodamine phalloidin (1:500, Fisher) for 1 h to visualize the actin cytoskeleton. 4ʹ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, UK) was added for a period of 10 min to visualize nuclei. Images were recorded using a Leica DM2500 fluorescence microscope (Leica, Cambridge, UK). Scale measurements were generated using ImageJ software.

Expression of COLI, COLII, and ACAN was determined in cell culture using specific antibodies (Sigma-Aldrich, Madrid, Spain). The cells were cultured on poly-L-lysine-coated cover slides in chondrogenic culture medium for up to 6 wk as described above. Then, they were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 min. Once washed, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, and after three washes, they were incubated for 30 min with blocking solution (1% bovine serum albumin (BSA) and 1.1% Tween-20 in PBS). The cells were then incubated with the appropriate primary antibody (diluted in antibody diluent at 1 : 100 for COLI and ACAN and 1 : 500 for COLII) overnight at 4°C. After three washes, cells were incubated with 1 : 200 secondary anti-mouse (COLI and ACAN) or anti-rabbit (COLII) FITC-conjugated antibody (Sigma-Aldrich, Madrid, Spain). After the final washes, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the samples were analyzed using the Leica DM2500 fluorescence microscope (Leica, Wetzlar, Germany).

Protein expression of Type I Collagen, vimentin, FSP1, and pankeratin in cell cultures was determined using specific antibodies. Cells were cultured in 8-well Millicell glass (Merck, Germany) at a density of 2 × 10³ (A549 cells) or 8 × 10³ (NFs or CAFs) cells/well. Cells were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min at RT. Once washed with PBS, cells were permeabilized with 0.1% Triton X 100 in PBS for 5 min, and after three washes with PBS, they were incubated for 30 min with blocking solution (1% bovine serum albumin and 1.1% Tween 20 in PBS). Cells were then incubated with the appropriate monoclonal primary antibodies diluted in antibody diluent solution. The primary antibodies used were: Cytokeratin Pan-Alexa fluor (1:100 dilution; Invitrogen, USA), Type I Collagen (1:100 dilution; Merck, Germany), Vimentin (2D1; 1:500 dilution; Novus Biologicals, USA) and FSP1 (1:100 dilution; Novus Biologicals, USA). Cells were incubated with the primary antibody overnight at 4 °C and after three washes, they were incubated with the anti-mouse FITC-conjugated secondary antibody (Sigma-Aldrich, Madrid, Spain) diluted 1:200, except cytokeratin, which was carried out by direct immunofluorescence. After the final washes, nuclei were stained with DAPI and samples were analyzed with a Leica DM2500 fluorescence microscope (Leica, Wetzlar, Germany).