Purified VLPs were mixed with Fabs at a ratio of 1:120 (capsid to antibody). Complexes were viewed on an FEI Spirit 120-kV TEM by negative-stain electron microscopy to confirm Fab decoration of the VLPs prior to sample vitrification. Three microliters of the AAV8-Fab complex samples was applied to glow-discharged C-Flat (CF-2/2 to 4c-50; Protochips, Inc.) holey carbon grids, blotted, and vitrified in liquid ethane using a Vitrobot Mark 4 (FEI) at 95% humidity and 4°C. The vitrified grids were stored in liquid nitrogen until data collection. For cryo-EM data collection, grids were transferred into Gatan cryosample holders and placed into an FEI Tecnai TF20 cryoelectron microscope operating at 200 kV. The complex data were collected at a magnification of 67,050× with a 1.5- to 3.0-μm defocus range under low-dose conditions (∼20 electrons/Å2) using a Gatan UltraScan 4000 charge-coupled device camera at 1.81 Å/pixel.
Spirit 120 kv tem
Manufactured by Thermo Fisher Scientific
The Spirit 120-kV TEM is a transmission electron microscope capable of operating at an accelerating voltage of 120 kilovolts. It is designed to provide high-resolution imaging and analysis of samples at the nanoscale level.
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2 protocols using spirit 120 kv tem
1
Cryo-EM Visualization of AAV8-Fab Complexes
2
Immunogold Labeling Protocol for TEM
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For post-embedding immunogold labeling, 70 nm-thick sections from specimens were prepared as described above. All postembedding steps except for the incubation with primary antibodies were performed at room temperature. For double immunogold labeling, sections were incubated with primary antibodies overnight at 4 C in a humidified chamber. After washing, the binding of primary antibodies was visualized by incubating with secondary antibodies conjugated to 5 nm and 10 nm gold particles for 60 min. Then grids were washed and stained with 2% aqueous uranyl acetate. Images were captured using a Spirit 120 kV TEM (FEI Company).
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