Anti α smooth muscle actin α sma

CCl₄ (C112040) and olive oil (O108686) were purchased from Aladdin (Shanghai, China). The hematoxylin and eosin (H&E) staining kit (G1120), Sirius Red staining kit (G1471), and Masson’s trichrome staining kit (G1340) were purchased from Solarbio (Beijing, China). TRIzol reagent was purchased from Invitrogen (Thermo Fisher SCIENTIFIC, MA, USA). A Universal Two-Step Test Kit (PV-9000) was purchased from ZsBio (Beijing, China). The following antibodies were used: anti-TRPM8 (GTX54866, GeneTex, California, USA), anti-α-smooth muscle actin (α-SMA) (BM0002, Boster, Beijing, China), anti-collagen type I (COL1A1) (BA0325, Boster, Beijing, China), anti-cytokeratin 19 (CK19) (GB11197, Servicebio, Wuhan, China), anti-β-actin (60008-1-AP, Proteintech, Rosemont, USA), anti-F4/80 (28463-1-AP, Proteintech, Rosemont, USA), anti-S100A9 (14226-1-AP, Proteintech, Rosemont, USA), and anti-HNF4α (ab41898, Abcam, Cambridge, UK).

The immunofluorescence assay was performed as previously described (13 (link), 29 (link)). Human lung sections, rats’ lung sections (13 (link)), and HPASMCs were first stained with anti-α-smooth muscle actin (αSMA, Boster, Wuhan), anti-MBD2 (Abcam), and anti-Ki67 primary antibodies (Abcam), followed by staining with Alexa Fluor 594-labeled anti-mouse/rabbit, or Alexa Fluor 488-conjugated anti-rabbit/mouse antibodies (Invitrogen, Carlsbad, CA, USA), respectively. DAPI was used for staining the cell nuclei. A Pannoramic MIDI (3Dhistech, Budapest, Hungary) was used to scan and analyze the images. Mean fluorescence intensity was assessed by ImageJ software (NIH, Bethesda, MD).

Paraffin sections of the lung were stained with hematoxylin–eosin (HE), anti-α-smooth muscle actin (α-SMA) (1:100; Boster Biological Technology, Wuhan, China), anti-proliferating cell nuclear antigen (PCNA) (1:100; Proteintech Group), and anti-TRB3 (1:100; Proteintech Group).