Mouse monoclonal anti cd31 antibody

To increase the permeation of printed GelMA to the antibody, the constructs were incubated with cool IFPerm III^® solution, which consists of 2 mL of acetone, 1 mL of methanol, 0.5 mL of formalin, 0.5 mL of Tween 20, and 0.25 mL of Triton X (all purchased from Sigma-Aldrich), at 4 °C for 2 days. They were washed twice with PBS and desiccated. They were then incubated with anti-human myosin heavy chain (MHC) antibody [clone MF 20, 1:10, developmental studies hybridoma bank (DSHB)] at 4 °C for 2 days and washed twice with 1× PBS prior. The constructs were then incubated with an anti-human immunoglobulin-G antibody (Sigma-Aldrich) labeled with FIT-C (fluorescein-5-isothiocyanate, 1:50, Sigma-Aldrich) at 4 °C for 1 day. They were washed twice with PBS and counterstained with DAPI. Immunofluorescence was acquired under the fluorescent microscope (Axio Vert.A1, Carl Zeiss) with red, blue and green filters. For HUVECs, 1/100 dilution of mouse monoclonal anti-CD31 antibody (Thermo Fisher Scientific) in a total volume of 200 µL was stained overnight.

Isolated mouse corneas were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 1 h and washed with PBS. The whole corneas were blocked with 5% normal goat serum (NGS; Thermo Fisher) and 2% BSA and permeabilized with 0.15% saponin (Sigma-Aldrich) for 30 min at room temperature, then incubated with mouse monoclonal anti-CD31 antibody (Thermo Fisher) for 2 h. After washes, the samples were placed in secondary IgG-horseradish peroxidase conjugate antibody (Jackson ImmunoResearch) for one hour. The samples were washed, and signals were revealed by 3,3′-diaminobenzidine (DAB; Sigma-Aldrich) reduction and examined under brightfield microscopy (Carl Zeiss Axioplan 2). Whole mount pictures (n = 5 in each group) were obtained, and the proportion of vessel ingrowth from limbal vasculature was assessed by overlaying a radial plot of 120 lines (giving 3° separation slot). The length of vessel ingrowth along each line was recorded and presented as a radial chart. The changes in the vascularized area were compared between treatment and control groups. Separately, wholemount images after double immunostaining for CD31 and LYVE-1 (protocol in the following section) were imported to ImageJ, and the width of CD31+ and LYVE+ vessels was recorded at multiple sites (n = 50 for each cornea and total n = 5 corneas in each treatment group). Mean vessel widths were calculated.