Tecnai 12 spirit

Three skin specimens of each setting were processed for transmission electron microscopy.
Two millimeter biopsies were taken (a) from the center of the wound (at t_{0 h} and t_{20 h}), (b) directly at the edge of the wound (at t_{20 h}), and (c) in close proximity to the wound (at t_{20 h}, area with intact epidermis) and fixated in 4% formaldehyde (in 50 mM HEPES) for 24 hours at room temperature. The skin pieces were washed (50 mM HEPES) and incubated for 10 min in 50% ethanol at room temperature. All skin samples were then dehydrated on ice in 70, 100, 100% ethanol (10 min each step), infiltrated with a LR White-ethanol solution (equal volumes, 10 min.), and incubated again with pure LR White (2x15 min). Subsequently, the skin pieces were transferred to polyallomer centrifuge tubes (5x 20 mm, No.342630, Beckman Coulter, Inc. Brea, CA, USA), containing LR White with accelerator (5 μL mL^-1 monomer). The centrifugation tubes were capped with gelatin capsule and polymerized for 1 hour on ice, and then incubated at 60°C overnight. Sections of ~75 nm were made for TEM imaging at the ultramicrotome (EM UC7, Leica, Wetzlar, Germany), stained with a solution of 0.9% uranyl acetate and 0.1% methyl cellulose (w/v) for 10 min, and imaged in the transmission electron microscope (Tecnai 12 Spirit; FEI, Eindhoven, Netherlands).

Electron microscopy was performed at Brigham Young University in the Life Sciences Microscopy Lab using an FEI Tecnai 12 Spirit transmission electron microscope. To prepare the samples for imaging, 20 μl of high-titer phage lysate was placed on a 200-mesh copper carbon type-B electron microscope grid for one minute. The lysate was wicked away and the grids were stained for two minutes using 2% phosphotungstic acid (pH = 7). Residual liquid was wicked away and the grid was allowed to dry before being imaged. Phage structures in electron micrographs were measured using ImageJ
[54 ]. The average and standard deviation for each measurement was calculated from a minimum of three separate measurements.

For EM analysis, gB-698glyco was incubated with 1G2 Fab at a 1:1.5 molar ratio on ice for 1 h and resolved over a Superose 6 PC 3.2/30 column connected to an Akta Micro system (GE Lifesciences). For visualization, 5 μl of sample was incubated on a freshly discharged continuous carbon 400-mesh copper grid (Electron Microscopy Sciences) for 30 sec. Following incubation, the grid was moved through 5 × 75 μl droplets of a 2% (w/v) uranyl formate solution (SPI Supplies). Excess stain solution was blotted away and the grid was air-dried. EM images were collected on a Tecnai-12 Spirit (FEI) equipped with a LaB6 filament operated at 120 keV under low dose conditions using a 4 k × 4 k CCD camera (Gatan Inc.) at a nominal magnification of × 49,000 (2.15 Å per pixel). Particles were isolated from individual micrographs using the e2boxer.py procedures embedded into EMAN2 (ref. 53 (link)) and subjected to iterative two-dimensional reference-free image analysis using multivariate statistical analysis followed by multi-reference alignment in IMAGIC5 (ref. 54 (link)). To obtain a gB/1G2 three-dimensional reconstruction, ∼10,000 particles were refined against an initial model of HSV gB (PDB ID 2GUM) low pass filtered to a resolution of 60 Å using the EMAN2/Sparx software53 (link)55 (link) and fitted in Chimera56 (link).