Ht 1002a

Homogeneous fibrin gels were prepared to final fibrinogen concentrations of 2 and $4\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L}$ by combining human fibrinogen (FIB3, Enzyme Research Laboratories) and Alexa Fluor (AF) 488 or 546 conjugated human fibrinogen (F13191, Molecular Probes) at a 1:10 fluorescent to non-fluorescent fibrinogen ratio, $1\mu \mathrm{L}$ 2M ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$ , and $0.5\mathrm{U}$ thrombin (HT 1002A, Enzyme Research Laboratories) per mg fibrinogen in phosphate buffered saline (PBS). Immediately after mixing, the solution was pipetted into molds containing frames that enabled tensile testing (Fig. 1a–c) and allowed to polymerize for 5 minutes. Molds $(2.5\times 6.6\times 0.75$ mm) were designed in SolidWorks and 3D printed (Stratasys J750) with a photopolymer (VeroClear, Stratasys) that is similar to acrylic. Frames $(6\times 12\mathrm{m}\mathrm{m})$ were drawn in Adobe Illustrator and laser cut from $100\mu \mathrm{m}$ thick polyethylene terephthalate using a Speedy 360 laser cutter (Trotec). Polyethylene (PE) blocks (coarse grade, Bel-Art Products) were manually cut to approximately $1.5\mathrm{m}\mathrm{m}\times 2.0\mathrm{m}\mathrm{m}\times 1.5\mathrm{m}\mathrm{m}$ . To increase wettability of PE by the fibrinogen solution, the blocks were submerged in $70\mathrm{\%}$ ethanol for 5 minutes at room temperature (RT), submerged in $12.91\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L}$ fibrinogen solution for at least 1 hour, then glued to the laser-cut frames with cyanoacrylate adhesive.