Alexa fluor 488 goat anti rabbit or anti mouse igg secondary antibody

For surface molecular staining, obtained cells were incubated with APC‐conjugated CD45.2 antibody (#558702; BD Biosciences, San Jose, CA, USA), Percp‐Cy5.5‐conjugated CD3e antibody (#45‐0031‐82; Invitrogen, Carlsbad, CA, USA ), FITC‐conjugated CD4 antibody (#11‐0041‐82; Invitrogen), APC‐Cy7‐conjugated Ly‐6G antibody (#560600; BD), PE‐conjugated Siglec‐F antibody (#552126; BD), PE‐CY7‐conjugated CD31 (#561410; BD) and PE‐conjugated EpCAM (#563477; BD). For intracellular staining, the Intracellular Fixation &Permeabilization kit (#88‐8824‐00; Invitrogen Co, Carlsbad, CA, USA) was used. The antibodies used were as follows: PE‐Cy7‐conjugated IL‐4 antibody (#25‐7042‐41; Invitrogen), APC‐conjugated IFN‐γ antibody (#17‐7319‐41; Invitrogen) and PE‐conjugated IL‐17 antibody (#561020, BD). Primary antibodies for intracellular staining included α‐SMA (#7817; Abcam), TGF‐β1 (#92486; Abcam), DEK (#166624; Abcam), E‐cadherin (#40772; Abcam) and Vimentin (#8978; Abcam). After that, incubation with Alexa Fluor 488 goat anti‐rabbit or anti‐mouse IgG secondary antibody (Invitrogen, #1810918, #2018309) was performed. The CD45.2^‐CD31^‐EpCAM⁺ cells were gated for lung epithelial cells and analysed.

Lung sections and BEAS‐2B cells were prepared and incubated with the primary antibodies of NF‐κB (#207297, Abcam), α‐SMA (#7817, Abcam), E‐cadherin (#40772, Abcam) and Vimentin (#8978, Abcam) at 4°C overnight and then incubated with Alexa Fluor 488 goat anti‐rabbit or anti‐mouse IgG secondary antibody (Invitrogen, #1810918, #2018309) for 2 hours. The samples were further observed using Cytation™ 5 (BioTek Instruments, Inc., Winooski, VT, USA).