Autofluo quencher

Lung tissues, aseptically isolated on day 3 p.i., were fixed in 4% paraformaldehyde (PFA) (Shanghai Life iLab Biotech) for 24 h at 4 °C. Subsequently, the tissues were subjected to gradient dehydration using 15% and 30% sucrose solutions for another 24 h before being embedded in Tissue-Tek O.C.T. Compound (SAKURA, Baltimore, MD, USA) and sectioned into cryosections with a thickness of 8 μm. The lung sections were blocked with 10% goat serum for 1 h at room temperature and incubated with anti-F4/80 antibody (1:150 dilution, ab16911, abcam, Cambridge, England) overnight at 4 °C. Next, they were rewarmed for 1 h before being incubated with Goat Anti-Rat IgG H&L (Alexa Fluor 488) preadsorbed secondary antibody (1:300 dilution, ab150165, abcam) for 1 h at room temperature in the dark. This was followed by washing five times using PBS. Then, AutoFluoQuencher (APPLYGEN, Beijing, China) was added and incubated for 15 min. Slides were then sealed by adding DAPI Fluoromount-G (SouthernBiotech, UAB, Birmingham, AL, USA), and images were acquired using a fluorescent microscope (200×).

This experiment was performed as previously described [25 (link)]. After deparaffinization and rehydration, slices were treated with 0.1% Triton X-100 for 15 min (for CypA only) and incubated with 1% bovine serum albumin for 1 h at 37°C to eliminate nonspecific staining. Without washing, slices were incubated with CypA rabbit polyclonal antibody (1:50) and MMP-9 goat polyclonal antibody (1:100) at 4°C overnight. The sections were washed and incubated with the secondary fluorescein donkey anti-rabbit Dylight 488 (1:200, EarthOx, San Francisco, CA, USA) and donkey anti-goat-CY3 (1:200, Proteintech Group, Wuhan, China) antibodies at 37°C for 1 h. Furthermore, 4′,6-diamidino-2-phenylindole was used to stain the nuclei at 37°C for 5 min. Finally, the sections were washed, incubated with AutoFluo Quencher (Applygen, Beijing, China) for 30 min, and viewed by fluorescent microscopy (Leica, Wetzlar, Germany). A single primary antibody (CypA, CD147 and MMP-9) or PBS was used as the control.