Cryo holder

Protein A and protein G-conjugated superparamagnetic beads (Bio-Adembeads) were purchased from Ademtech. Before use, the beads were washed with a large volume of working buffer (PBS). The magnetic beads (10 μl) were incubated with anti-BVDV E2 mAb or anti-KDEL mAb (control) in a final volume of 200 μl, at 4°C for 30 min. The magnetic bead–protein A/G–antibody complexes were sedimented with a magnet. The pellets were washed three times to remove excess antibody. Beads coated with antibody were incubated with BVDV at room temperature for 30 min (final volume 100 μl). After sedimentation with the magnet, the pellet was washed in order to remove unbound particles. Cryo-TEM lacey carbon cupper grids were then prepared with this suspension, without any further treatment according to the protocol described above. Specimens were maintained at a temperature of approximately -170°C, using a cryo holder (Gatan, CA, USA) and observed with a FEI Tecnai12 electron microscope operating at 120 kV and at a nominal magnification of 30,000 x under low-dose conditions. Images were recorded with a 4k x 4k slow-scan CCD camera (FEI).

Cryo-TEM samples of isolated membranes obtained from glioblastoma multiform U87-MG cell line, Ct-NE and CM-NESoSome 0.25 wt% oil were prepared by Plunge freezing technique by using the Vitrobot Mark IV (Fei Company). Overall, 3 μL of each specimen were applied on 200 mesh quantifoil cupper grid in Vitrobot chamber and subsequently reduced in volume by blotting for 1 s with filter paper to yield a final thin film about 100–200 nm in thickness. To prevent sample evaporation, the Vitrobot was settled to 95% humidity and 4 °C with a waiting time of 60 s before plunging in liquid propane. After grid transfer in liquid nitrogen, each sample was mounted on Gatan Cryo holder, then observed by transmission electron microscope TECNAI G2-20 (Fei Company, Hillsboro, OR, USA) equipped by Gatan CCD camera 2HS, in Cryo mode. The imaging was performed in low dose mode, at 200 KV in a range of magnification from 20,000 to 50,000.

Samples of NPs were prepared to be at a mass percentage of 1% particles in PBS and the Cryo-TEM specimens were prepared in a controlled environment vitrification system (CEVS). Cryogenic transmission electron microscopy (Cryo-TEM) imaging was performed using a FEI Talos 200C, FEG-equipped Cryo-dedicated high-resolution transmission electron microscope (TEM and STEM), operated at an accelerating voltage of 120 kV. Specimens were transferred into an Oxford CT-3500 Cryo-holder (Philips) or a Gatan 626DH (FEI) Cryo-holder, and equilibrated below −178 °C. Specimens were examined using a low-dose imaging procedure to minimize electron-beam radiation damage. Images were recorded digitally by a Gatan Multiscan 791 cooled CCD camera (Philips CM 120), or a Gatan US 1000 high-resolution CCD camera (Tecnai T12 G2), using DigitalMicrograph software.