Formvar carbon coated 400 mesh copper grids

NFP (10 × 10⁻⁶m, 20 μL) was dropped onto formvar/carbon-coated 400 mesh copper grids (Electron Microscopy Sciences, Hatfield, PA) and an excess sample was removed with filter paper. After uranyl formate staining (0.5% v/v, 20 μL), the grids were examined under TEM (JEOL JEM-1400 LaB₆ TEM operating at 120 kV). For the TEM analysis of the tumor sections, tissues were first fixed in modified Karmovsky’s fix solution (2.5% glutaraldehyde, 4% paraformaldehyde, and 0.02% picric acid in 0.1 m buffer) and then in reduced osmium tetroxide (1% OsO⁴⁻ and 1.5% K-ferricyanide (aqueous). Following dehydration, the samples were embedded in an Epon analog resin (Embed812) and ultrathin sections (65 nm) were cut using a Diatome diamond-knife on Ultracut T ultramicrotone (Leica Microsystems, Wetzlar, Germany). The sections were contrasted with lead citrate and observed on the JEM 1400 electron microscope.

Peptide formulations were diluted to 0.2 mM total peptide in PBS and vigorously
vortexed. Five microliters of 0.2 mM peptide nanofibers was deposited onto Formvar/carbon
coated 400 mesh copper grids (Electron Microscopy Sciences), washed, stained with
1% w/v uranyl acetate in water, and dried. Samples were imaged on an FEI Tecnai
Spirit TEM.

Nanofiber morphology was characterized by transmission electron microscopy (TEM). Nanofiber formulations were diluted to 0.2 mM with PBS and deposited onto Formvar/carbon-coated 400-mesh copper grids (Electron Microscopy Sciences). After 1 min incubation, the grids were washed with water and stained with 1% w/v uranyl acetate in water for 1 min. Excessive liquid was then wicked away with filter paper, and grids were air-dried before imaging on an FEI Tecnai F30 TEM.

20 μL of TMV-TBP_T25-biotin was place on Formvar/carbon-coated 400 mesh copper grids (Electron Microscopy Science) for 30 min. The grid was washed once with 10 mM KP buffer (pH 7), and blocked with 1% (w/v) BSA with 0.1% (v/v) Tween-20 for 30 min followed by equilibration in 0.1% (w/v) BSA for 5 min. The grid was then placed onto a droplet of goat anti-biotin (1:5 dilution, 10 nm-sized gold labels, Aurion) and incubated for 1 h. Following the incubation, the grid was washed once with PBST (PBS containing 0.1% (v/v) Tween-20) for 3 mins, then washed thrice with distilled water (5 min each wash). Lastly, the grid was stained with 2% (w/v) uranyl acetate (Fisher Scientific) for 2 min. Excess solution was blotted with filter paper. The grid was imaged with a JOEL 1400 transmission microscope at 80 kV.

20 μL of CPMV-CBP-biotin was place on Formvar/carbon-coated 400 mesh copper grids (Electron Microscopy Science) for 20 min. The grid was washed once with 10 mM KP buffer, pH 7, and blocked with 1% (w/v) BSA in 0.1% (v/v) Tween-20 for 30 min followed by equilibration in 0.1% (w/v) BSA in 0.1% (v/v) Tween-20 for 5 min. Later, the grid was then placed onto 20 μL of streptavidin-conjugated gold nanoparticles (5 nm sized gold, Nanocs) and incubated for 2 h. Following the incubation, the grid was washed four times with 0.01% TBST (3 min each wash), twice with 10 mM KP buffer (3 min each wash), and thrice with distilled water (5 min each wash). Lastly, the grid was stained with 2% (w/v) uranyl acetate (Fisher Scientific) for 2 min. Excess solution was blotted with filter paper. The grid was imaged with a FEI Tecnai G2 Spirit transmission microscope at 80 kV.