Af 488 goat anti mouse

The seeded fibroblasts were recorded after 14 days. The fluorescence was generated by 30-minute incubation with 2 μM calcein AM in PBS.
SMCs in cell-seeded fibers at day 3 and day 7 were fixed in 4% paraformaldehyde for 30 min in room temperature. Following fixation, fibers were washed 3x with PBS, permeabilized with 0.1% Triton X-100, and blocked using 2% BSA in PBS for 1 h at 4°C. After washing 3x at room temperature in PBS and immunohistochemistry labeling on fibers and control (chitosan film) samples was performed, applying primary antibody against alpha-smooth muscle actin (monoclonal mouse anti-human), at 1 : 100 dilutions in PBS/BSA/buffer at room temperature for 2 h. After washing 3 times for 10 min in PBS/BSA buffer, secondary antibodies (AF 488 goat anti-mouse, Invitrogen) were applied at 1 : 200 dilutions in PBS/BSA buffer at room temperature for 1 h. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and PCL fibers were then imaged via confocal microscopy (Leica, Wetzlar, Germany).

Immunofluorescence was performed on archival frozen sections of 3 patients with active CD, 3 with inactive CD and 3 controls. Samples were embedded in a cryostat mounting medium [Neg–50 Frozen Section Medium, Thermo Scientific], snap frozen and stored at −80°C. Sections 6 µm thick were mounted onto superfrost plus glass slides [Thermo Scientific] and fixed in 4% paraformaldehyde [PFA] for 10 min at 4°C. Slides were washed three times with PBS and treated with 0.1% Triton X-100 for 20 min at room temperature [RT]. Blocking was performed with a 10% normal goat serum PBS solution for 1 h at RT. Slides were then incubated overnight at 4°C with mouse anti-human CD3 (1:100, M3070 from Spring Bioscence); or hamster anti-human CD11c (1:75, MA11C5 from Invitrogen); and rabbit anti-human/mouse ADAR1 (1:100, sc-73408 from Santa Cruz Biotechnology). After washing three times with PBS, slides were incubated for 1 h at RT with specific secondary antibodies AF 488 goat anti-mouse (A11017), AF 568 goat anti-rabbit (A11011), AF 568 goat anti-hamster (A21112) all 1:2000 from Invitrogen. Coverslips were mounted on glass slides using ProLong Gold antifade reagent with DAPI [Invitrogen] to counterstain the DNA. Images were acquired on a Leica DMI 4000 B fluorescence microscope [Leica].