Dab stain

Sections were deparaffinized and treated with proteinase K working solution at 37 °C for 20 min. Each specimen was then exposed to a mixture 5 µL TdT+45 µL fluorescein-labeled dUTP, incubated for 1 h at 37 °C and then washed with PBS three times. Next, 50 µL of converter-POD was added to the mixture for 30 min at 37 °C and then sections were again washed with PBS three times. The reaction was visualized with DAB stain (P0203, Beyotime Biotechnology, Shanghai, China) and counterstained with hematoxylin and the progress of the reaction observed with the aid of a microscope (Olympus IX73, Japan).

The lung slides were prepared following previously described protocols [28 (link)]. The lung sections were incubated with the primary antibodies overnight at 4 °C, then the secondary antibody biotinylated anti-rabbit IgG (1:100, Beyotime, Guangzhou, China) was incubated for 60 min at 37 °C. Followed by incubating with SABC (1:100, Beyotime, Guangzhou, China), and then visualized by DAB stain (Beyotime, Guangzhou, China). Cell nuclei were stained with hematoxylin.

Mice or IPF paraffin-embedded tissue sections were deparaffinized and then permeabilized with 0.3% Triton X-100 (5 min), followed by blocking with endogenous peroxidase blocking buffer (10 min) (Beyotime), antigen retrieval was carried out by heat mediation in a citrate buffer solution (pH = 6) (20 min). Sections were closed in a blocking buffer (60 min) (Beyotime) and immunostained with anti-α-SMA (1:100; Abcam, ab240654), anti-MMP19 (1:100; Affinity, AF0215), anti-ET1 (1:100; Abcam, ab2786), anti-SDF1 (1:100; Affinity, AF5279), anti-collagen I (1:100; Abcam, ab138492), anti-collagen IV (1:100; Abcam, ab6586), anti-CXCR4 (1:100; Abcam, ab181020), anti-EMR1 (1:100; Affinity, DF2789) overnight at 4 °C, followed by incubation for 1 h at RT with biotin-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies followed by incubating with SABC (1:100; Beyotime), and then visualized by DAB stain (Beyotime). Cell nuclei were labeled with hematoxylin.