Mouse or human skin was dehydrated, paraffin embedded, and sectioned (4 µm). Sections were rehydrated, quenched with 3% hydrogen peroxide, incubated in citric buffer (VectorLabs) for antigen retrieval, and blocked with Avidin/Biotin Blocking System (VectorLabs) and then 5% normal goat serum.
For double immunohistochemistry staining for CFIm25 and α-SMA, sections were incubated with antibodies for CFIm25 (1:400; Proteintech) overnight at 4°C and then with biotinylated anti-Rabbit antibodies (1:1,000; VectorLabs) for 1 h at room temperature and ABC Elite streptavidin reagents for 30 min at room temperature. Slides were then developed with 3,3-diaminobenzidine (Sigma-Aldrich). After development, the slides were incubated with mouse anti-α-SMA antibodies (1:1,000; Sigma-Aldrich) overnight at 4°C, anti-mouse secondary antibodies (1:1,000; VectorLabs) and alkaline phosphatase ABC Elite streptavidin reagents and developed using Vector Red Substrate (VectorLabs).
For CFIm25/α-SMA immunofluorescence dual staining, skin sections were first stained with CFIm25 and developed with Vecto Red Substrate. The sections were then blocked with normal horse serum and incubated with anti-α-SMA antibodies and Alexa Fluor 488 Goat Anti-Mouse IgG (Life Technologies). Slides were finally mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies).
For double immunohistochemistry staining for CFIm25 and α-SMA, sections were incubated with antibodies for CFIm25 (1:400; Proteintech) overnight at 4°C and then with biotinylated anti-Rabbit antibodies (1:1,000; VectorLabs) for 1 h at room temperature and ABC Elite streptavidin reagents for 30 min at room temperature. Slides were then developed with 3,3-diaminobenzidine (Sigma-Aldrich). After development, the slides were incubated with mouse anti-α-SMA antibodies (1:1,000; Sigma-Aldrich) overnight at 4°C, anti-mouse secondary antibodies (1:1,000; VectorLabs) and alkaline phosphatase ABC Elite streptavidin reagents and developed using Vector Red Substrate (VectorLabs).
For CFIm25/α-SMA immunofluorescence dual staining, skin sections were first stained with CFIm25 and developed with Vecto Red Substrate. The sections were then blocked with normal horse serum and incubated with anti-α-SMA antibodies and Alexa Fluor 488 Goat Anti-Mouse IgG (Life Technologies). Slides were finally mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies).