G7402

For immunohistochemical analyses specimens were placed in ice-cold isopentane, stored at -80°C and followed by standardized procedures to produce paraffin blocks and paraffin sections of 2 μm thickness. Immunohistochemistry was performed according to standardized methods in a fully automated manner (Dako Autostainer plus, Dako, Germany) by use of a 1:400 dilution for the first rabbit anti-ChAT antibody (Abcam ab68779, UK; second ab: Envision Flex+rabbit, Dako K8019, Germany). Histology was evaluated and documented using a Keyence BZ-9000E Hs all-in-one microscope (Keyence, Germany). For immunogold transmission electron microscopy specimens were fixed in buffered formaldehyde (3.7%) for 2 h at room temperature and stored overnight in phosphate-buffered saline at 4°C. Fixed specimens were embedded in epoxide, followed by the use of a standard protocol with application of the first antibody (ab68779) in a dilution of 1:100 and the second gold-labeled anti-rabbit antibody (Sigma Aldrich, G7402) in a dilution of 1:20. For scanning electron microscopy specimens were fixed in 2.5vol% glutaraldehyde (6 hours at room temperature). The analyses and photo-documentation were performed by using a transmission electron microscope (JEM-1400, Jeol, Tokio, Japan). Negative control experiments without the primary antibody were always carried out in parallel.

Samples for transmission electron microscopy were prepared using traditional chemical fixation [65 (link)]. A total of 3 to 4 leaves were fixed in 2% (v/v) glutaraldehyde and 0.5% (w/v) paraformaldehyde in 50 mM PBS for 2 hours at room temperature (RT), rinsed 3 times in 50 mM PBS, dehydrated, infiltrated, and embedded in LR White resin (Sigma-Aldrich). Resin blocks were polymerized at 48°C for 48 hours and serially sectioned ultrathin (approximately 80-nm thick). Ultrathin sections were blocked and subsequently incubated in a moist chamber with primary antibody diluted 1:150 (anti-GFP; Abcam Ab290) 2 hours at RT. Secondary antibody that conjugated to 10-nm gold particles (Sigma-Aldrich; G7402) were diluted 1:50 with PBS/BSA and incubated for 1 hour at RT. Sections were extensively washed, stained with uranyl acetate, and examined with FEI Tecnai Spirit D1297 transmission electron microscope (FEI, Hillsboro, OR, USA).

Cells were first incubated with PEDVs at a high MOI of 1,000 for 30 min at 4°C, then transferred to 37°C, fixed with immune electron microscopy fixative (G1124; Servicebio) for 2 h at room temperature, and finally collected. Double-label immunoelectron microscopy was performed with standard procedures. Briefly, the collected cell precipitation was resuspended and washed twice with precooled 0.1 M PBS (pH 7.4). After the supernatant was removed, samples were processed with dehydrating, resin penetrating, embedding and polymerizing steps. Sample resin blocks were sliced into 90-nm ultrathin cryosections using a Leica FC7 ultramicrotome and collected onto the 150-mesh nickel grids with Formvar film for immunogold labeling. The nickel grids were incubated with 1:100 dilution of rabbit anti-clathrin light chain antibody (ab150658; Abcam) and 1:50 dilution of mouse anti-caveolin-1 antibody (ab17052; Abcam) overnight at 4°C. The nickel grids were rinsed with PBS 3 times at 5 min each and then were incubated with a 1:50 dilution of gold-conjugated goat anti-rabbit IgG (G7402, 10 nm; Sigma) and 1:100 dilution of gold-conjugated goat anti-mouse IgG (115-185-146, 4 nm; Jackson) for 1 h at 37°C. The grids were washed and stained with 2% uranyl acetate. Finally, the sections were examined with a transmission electron microscope (HT7800; Hitachi).