Alexa fluor 647 goat anti rabbit secondary antibody

The cells on fibrin network were fixed with 4% paraformaldehyde (Beyotime, China) and permeated with 0.2% TritonX‐100 (Sigma‐Aldrich, USA). Cells were blocked with 1% BSA in PBS and incubated with Integrin β3 (Affinity, AF6086, 1:100), Zyxin (Abcam, ab109316, 1:500), c‐Src (Proteintech, 60315‐1‐Ig, 1:100), and CCR7 (Abcam, ab32527, 1:100) antibody overnight at 4 °C. After incubation, the cells were washed and incubated with Alexa Fluor 647 goat anti‐rabbit secondary antibody (Beyotime, China) and Alexa Fluor 594 goat anti‐mouse secondary antibody (Emarbio, China) at 20 °C for 1 h. Then the cells were washed and stained for F‐actin (Beyotime, China) and nuclei with DAPI staining solution (Beyotime, China). Fluorescent images were observed with a confocal laser scanning microscope (FV3000 Olympus, Japan).

The extracted total RNA was detected by Nanodrop (Thermo Fisher Scientific, USA) and then converted to complementary DNA by PrimeScript RT Master Mix (Takara, Japan). Equivalent cDNA samples were analyzed by RT-qPCR analysis using Hieff qPCR SYBR Green Master Mix (Yeasen, China) on an ABI 2-step system (Applied Biosystems, USA). Primer sequences were shown in the Supplementary Materials (Tables S1 and S2). The obtained samples were calculated based on the 2^−ΔΔCt method referring to glyceraldehyde-3-phosphate dehydrogenase. Macrophages were stained by 4′,6-diamidino-2-phenylindole simultaneously with integrin β₂ (1:100; Proteintech), Zyxin (1:500; Abcam), Src (1:100; Proteintech), LC3 (1:100; Cell Signaling Technology), and CCR7 (1:100; Abcam). Fibroblasts were stained by actin, 4′,6-diamidino-2-phenylindole, α-SMA (1:100; Affbiotech), and collagen I (1:100; Abcam) antibody. Alexa Fluor 594 goat anti-mouse secondary antibody (Emarbio, China) and Alexa Fluor 647 goat anti-rabbit secondary antibody (Beyotime, China) were used for different purposes.