Mouse anti α sma

Briefly, after deparaffinization, hydration, and antigen retrieval, the skin tissue sections were blocked with 5% BSA at room temperature for 1 h and then incubated with the primary antibodies rabbit anti-CD31(1:300, Servicebio, Wuhan, China) and mouse anti-α-SMA (1:200, Servicebio) (dissolved in 1% BSA) at 4 °C overnight. After washing with PBS, the sections were incubated with Cy3-conjugated goat anti-rabbit IgG (1:300, Servicebio) and Alexa Flour 488-conjugated goat anti-mouse IgG (1:400, Servicebio) at room temperature for 1 h. Afterward, DAPI (Servicebio) was added dropwise and incubated for 10 min. Finally, sections were captured by the virtual slide microscope (VS120-S6-W, OLYMPUS), and the analysis of CD31 and α-SMA positive staining cells was quantified using ImageJ software.

Lung tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin. Then, 5 µm thick sections were deparaffinized in serial solutions of xylene, gradient ethanol, and water, followed by antigen retrieval by steaming in 1 mM EDTA for 5 min in a pressure cooker. The lung sections were washed with PBS buffer and blocked with 5% BSA for 30 min. Next, blocked samples were incubated with mouse anti-α-SMA (1:200; Servicebio, Wuhan, China) or anti-TREK–1 antibody (1:200, Alomone Labs, Jerusalem, Israel) and F4/80 antibody (1:200, proteintech, Wuhan, China) overnight at 4 °C followed by exposure to staining with corresponding secondary antibodies for 1 h at room temperature. Slides were then counterstained with nuclear dye DAPI and mounted. Images were acquired on a fluorescence microscopic imaging system (Leica, Weztlar, Germany).