Em ace600 high vacuum sputter coater

Negative-stained EM samples were prepared by diluting purified augmin to 150 nM in CSF-XB and pipetting 3 µl onto glow-discharged (15 mA, 25-30 secs) carbon film, 400 mesh Cu grids (Electron Microscopy Sciences), staining with 0.75% uranyl acetate solution. Negative-stain EM data was collected at 94,000x magnification (1.56 Å/pixel) with single-tilt using a Talos F200X Transmission Electron Microscope equipped with a 4k x 4k Ceta 16 M CMOS camera.
Cryo-EM grids were prepared similarly using undiluted, purified augmin. 0.05% NP-40 was added to augmin prior to applying to grids. Here, 3 µl of sample was applied to glow-discharged (10 mA, 8 sec) Quantifoil holey carbon R 1.2/1.3 400 mesh grids coated with a home-made thin carbon film (~5 nm thickness) using Leica EM ACE600 High Vacuum Sputter Coater. The grids were flash frozen in liquid ethane using a FEI Vitrobot Mark IV (Thermo Scientific) plunge freezer, using a blot force of 0 and with a 4.5 sec blot time. Cryo-EM data were collected using the Titan Krios microscopes at either Washington University in St. Louis (WUSTL) or Case Western Reserve University (CWRU). The data collection parameters are listed in Supplementary Table 1.

Tensile tests were carried out on ISO 527-1A dog-bone specimens using an MTS Criterion model 43 universal testing machine (MTS Systems Corporation, Eden Prairie, MN, USA) at a crosshead speed of 10 mm/min, equipped with a 10 kN load cell and interfaced with a computer running MTS Elite Software. Tests were carried out after two days after the injection-molding process and, during this time, the specimens were stored in a dry keeper at controlled atmosphere (room temperature and 50% of relative humidity). At least five specimens were tested for each blend composition.
Charpy impact tests were performed on notched samples. The V-notch in the center of the specimens was made by a manual V-notch cutter (45° V-notch; depth: 2 mm). For the impact tests, an Instron CEAST 9050 machine (INSTRON, Canton, MA, USA) was used. Five samples of each blend composition were tested at room temperature; in this case, the tests were carried out after two days after their injection molding.
Blend morphological characterization was performed on cryo-fractured Charpy samples by FEI Quanta 450 FEG scanning electron microscopy (SEM; Thermofisher Scientific, Waltham, MA, USA). To avoid charge build-up, the samples were sputtered beforehand (with an LEICA EM ACE 600 High Vacuum Sputter Coater, Wetzlar, Germany) with a thin surface layer of platinum.

The specimen for SEM imaging were prepared using different sample preparation methods to obtain aerogels. GrowDex and agarose samples were plunge-freezed in liquid propane and subsequently lyophilized. The alginate sample was prepared using a supercritical carbon dioxide drying method. Matrigel, ovomucin, collagen and alginate-RGD samples were prepared using glutaraldehyde (3.5%) fixation for overnight. After washing the excess fixative with water, the specimen was dehydrated with a series of water/ethanol (70/30, 50/50, 30/70, 10/90 and 0/100 v/v) mixture by incubating 10 min in each solution. Finally, samples were chemically dried with hexamethyldisilazane (HMDS) by incubating in each solution for 10 min in 1:1 (v/v) ethanol: HMDS and 2 x HMDS.
SEM images were acquired with a Zeiss Sigma VP scanning electron microscope with an acceleration voltage of 1.0 to 1.5 kV. The aerogel sample was placed on the carbon tape and sputter-coated with Au/Pd with Emitech K950X/K350 or a Leica EM ACE600 high vacuum sputter coater. Sample preparation methods, coatings, and acceleration voltages for each matrix appear in Supplementary Table 1a.