Ab232784

Firstly, the prepared CAFs and NFs were observed under a microscope. Subsequently, the CAFs and NFs were seeded into a 24-well plate (at a density of 5000 cells/well), fixed with 4% paraformaldehyde (PFA) for 15 min, treated with 0.5% Triton-X-100 for 20 min, and blocked with 3% bovine serum albumin (BSA) for 1 h. Next, the cells were subjected to incubation with antibodies α-SMA (ab232784, Abcam, Cambridge, MA, USA), FAP (PA5–99313, Thermo Fisher Scientific, Waltham, MA, USA), or vimentin (ab92547, Abcam) at 4 °C overnight. The following day, the cells were incubated with the secondary antibody goat anti-rabbit IgG (ab6721, Abcam) for 45 min in conditions devoid of light. After removing the secondary antibody solution, the cells were rinsed with PBS thrice (5 min), incubated with 2,4-diaminobutyric acid (DAB) in dark conditions for 5 min, rinsed with PBS (2 × 5 min), counterstained with hematoxylin for 1 min, and sealed with neutral gum. Afterwards, the cells were observed under an optical microscope (CKX51, Olympus, Tokyo, Japan). Finally, the expression patterns of α-SMA, FAP, and vimentin were determined using reverse transcription quantitative polymerase chain reaction (RT-qPCR).

First, the mice lung tissues were lysed with RIPA lysis buffer. Bicinchoninic acid (BCA) was used to detect total protein. Then, the same amount of protein was separated by 10% SDS-PAGE, and the protein band was transferred to the polyvinylidene fluoride (PVDF) membrane. After washing with TBST buffer, it was blocked with 5% skimmed milk for 2 h, incubated with primary antibody α-SMA (ab232784, Abcam, UK), collagen I (ab270993, Abcam, UK), HMGB1 (ab18256, Abcam, UK), p-NF-κBp65 (ab 183559, Abcam, UK), ß-actin (ab8226, Abcam, UK) at 4°C overnight, and washed 3 times with PBS. Then, it was incubated with HRP-conjugated anti-rabbit IgG secondary antibody for 2 h at room temperature. Finally, an enhanced chemiluminescence (ECL) solution (Millipore, USA) was added for development. ImageJ 1.50 was used for quantitative analysis.