Ab92572

Immunohistochemistry was performed to test whether fibrinogen and collagen type I were present in the adhesion lesions and exudates. Tissue specimens were fixed for 24 h in IHC Zinc fixative solution (BD Biosciences) and embedded in paraffin wax before sectioning. Sections of 4 µm thickness for each specimen were prepared on peeling prevention coated slide glass (Matsunami Glass Ind.) and stained with hematoxylin and eosin (H&E). For immunohistochemistry, the sections were preincubated with serum-free protein block (Agilent Technologies) for 30 min at room temperature and primary antibodies for fibrinogen (ab92572, Abcam) and collagen type I (F56, Kyowa Pharma Chemical Co. Ltd.) diluted in PBS were added to each slide overnight at 4 °C. Secondary antibody staining was performed using the Histofine Simple Stain MAX-PO (Nichirei) according to the manufacturer’s instructions. The staining was visualized under a Nikon Eclipse TS-100 microscope (Nikon, Tokyo, Japan).

A random selection of some sections from each group were rewarmed at 37 °C for 30 min, wetted with PBS for 3 min, then permeabilized with 0.2% Triton X-100 (Sigma, USA) for 10 min at room temperature, washed three times with PBS, and incubated in blocking agent (10% donkey serum; Solarbio) to block nonspecific binding. Sections were incubated with one of the primary antibody solutions—anti-fibrinogen (ab92572, 1:200; Abcam), anti-CTGF (ab6992, 1:200; Abcam), or anti-vWF (ab6994, 1:200; Abcam)—overnight at 4 °C, rinsed thoroughly with PBS three times to remove unbound primary antibody, and then incubated with a secondary antibody of donkey anti-rabbit IgG H&L (Alexa Fluor® 594) (ab150076, 1:500; Abcam) for 1 h at 37 °C. Nuclei were stained with Hoechst 33342 (1:1000; Sigma) for 5 min. The sections were disposed with Enhanced Antifade Mounting Medium (Leagene, China) and observed by laser scanning confocal microscopy (Nikon A1R, Japan).

Western blotting was conducted in accordance with previous work (24 (link)). The primary antibodies against FGA (ab92572, 1:2000, Abcam, USA), integrin αν (#4711, 1:1000, Cell Signaling Technology, USA), integrin β3 (#13166, 1:1000, Cell Signaling Technology, USA), AKT (#4685, 1:1000, Cell Signaling Technology, USA), P-AKT (#4060, 1:1000, Cell Signaling Technology, USA), ERK (#4695, 1:1000, Cell Signaling Technology, USA), P-ERK (#4370, 1:1000, Cell Signaling Technology, USA), FAK (#13009, 1:1000, Cell Signaling Technology, USA), P-FAK (#3282, 1:1000, Cell Signaling Technology, USA), matrix metalloproteinase-2 (MMP-2) (ab97779, 1:1000, Abcam, USA) and MMP-9 (ab137867, 1:1000, Abcam, USA) were incubated at 4°C overnight. The target proteins were visualized by the chemiluminescent gel imaging system (Bio-Rad, USA) after incubation with a secondary antibody (7074, 1:3000, Cell Signaling Technology, USA). Each experiment was tested in three replicates.