Anti fibronectin antibody

Samples were then sectioned in 5 μm thickness. Sections and RAW264.7 cells cultured on coverslips for immunofluorescence were fixed with acetone, washed with PBS for three times, and blocked for 1 h. The sections were then incubated at 4 °C overnight with primary antibodies, including anti-fibronectin antibody (1:200, No. ab23750, Abcam, Cambridge, UK), anti-collagen 1 antibody (1:200, No. ab34710, Abcam, Cambridge, UK), and anti-α-smooth muscle actin antibody (1:200, No. A5228, Sigma, St. Louis, MO). The antibodies for double staining is anti-CD45 antibody (1:100, No. 610265, BD, Franklin Lakes, NJ), anti-F4/80 antibody (1:100, No. MCA497GA, Bio-Rad, Hercules, CA), anti-CD206 antibody (1:100, No. MCA2235, Bio-Rad, Hercules, CA), or anti-αSMA antibody (1:100, No. ab5694, Abcam, Waltham, MA). The immunofluorescence was detected using Alexa Fluor 488 antibody (Invitrogen, Carlsbad, CA) or Alexa Fluor 647 antibody (Invitrogen, Carlsbad, CA). Images were obtained on a fluorescence microscope equipped with a digital camera (Leica, Wetzlar, Germany) or confocal microscope (LSM 880, Zeiss, Jena, Germany). Measurement of the fluorescence staining area and count of positive cells was performed using ImageJ (NIH Image J system, Bethesda, MD).

Fibronectin functionality was evaluated through antibody binding. Printed GO and control substrates were blocked with 5% (v/v) BSA (Sigma-Aldrich) in PBS solution at room temperature (20 min). The blocking solution was removed and the monoclonal mouse anti-fibronectin antibody in PBS (10 μg/ml, abcam) was added. The samples were incubated at room temperature for 90 min. Following incubation, samples were washed 3 times with PBS and subsequently exposed to the anti-mouse antibody-Cy5 conjugates (7.5 μg/ml, λ_Exc = 649 nm, λ_Em = 666 nm, Invitrogen) at room temperature in dark conditions for 60 min. Samples were washed twice with PBS before being stored at 4 °C and submerged in PBS. The secondary antibody position was assessed using fluorescent microscopy.

Far-WB analysis was carried out according to a previous procedure (Chen et al., 2019 (link)). In brief, approximately 20 μg of Mhp Eno, Mp Eno and Mb Eno was transferred to a PVDF membrane. BSA was used instead of mycoplasma enolases as a negative control. After blocking with skimmed milk, the membrane was incubated with 5 μg/ml plasminogen or fibronectin (Roche). Then anti-plasminogen or anti-fibronectin antibody (Abcam; 1 μg/ml) was used to interact with the membrane as the primary antibody. Finally, horseradish peroxidase (HRP)-conjugated anti-IgG (Boster; 1:5,000 dilution) and electrochemiluminescence kits (Thermo Scientific) were used to detect proteins in the membrane.

Cell lysate combinations of RIPA with PIC2 and PPI were used to form the lysis buffer, and a reaction time of 30 min in an ice bottle was allocated. The protein concentration in each cell lysate was then measured using a commercial BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). In the Western blot analysis, an SDS-polyacrylamide gel electrophoresis system was used. An anti-GAPDH antibody (Cell Signaling, Danvers, MA, USA), anti-fibronectin antibody (Abcam, Cambridge, England, UK), anti-vimentin antibody (cell signaling), anti-E-cadherin antibody (BD Biosciences, Franklin Lakes, NJ, USA), anti-ZO-1 antibody (BD Biosciences), anti-PCNA antibody (Cell Signaling), anti-protein kinase B (AKT) antibody (Cell Signaling), anti-phospho-AKT (P-AKT) antibody (Cell Signaling), anti-α7-nAChR antibody (Abcam), and anti-SNCG antibody (Cell Signaling) were used for probing.

Liver tissues were fixed with 4% formaldehyde and sectioned for H&E and Sirius Red staining. Liver fibrosis was assessed according to the METAVIR scale (F0, no liver fibrosis; F1, portal fibrosis; F2, periportal fibrosis; F3, bridging fibrosis; F4, liver cirrhosis) [20 (link)]. After being deparaffinized and serially dehydrated, the sections were treated with hydrogen peroxide for 30 minutes and then incubated with primary antibody overnight at 4°C. The primary antibodies used for immunohistochemical staining were anti-angiotensin-converting enzyme 1 (ACE1) antibody (1 : 200, Abcam, MA, USA) and anti-fibronectin antibody (1 : 2000, Abcam, MA, USA). After treatment with a biotinylated secondary antibody for 30 minutes at 37°C, the positive areas were stained with diaminobenzidine (DAB) and the nuclei were counterstained with hematoxylin. Immunohistochemical analysis was performed with Image-Pro Plus 6.0 software.