Talos l120

High-pressure freezing, freeze substitution, and sectioning were all performed as previously described in [24 (link),109 (link)–111 (link)]. AIY Zone 2 was identified based on the anatomical landmarks described in the original C. elegans connectome and recent Zone 2 reconstruction [24 (link),31 (link)].
For immuno-EM, sections of 50 nm were collected on nickel slot grids covered with Formvar (EMS). Grids were incubated at 20°C on 50 μl droplets of 0.05 M glycine PBS for 5 min, 1% BSA and 1% CWFS gelatin in PBS for 20 min, anti-GFP rabbit polyclonal (1:20 in 0.3% BSA and 0.3% CWFS gelatin in PBS, ab6556 Abcam) overnight at 4°C and then 60 min at 20°C, 6 PBS washes over 30 min, Protein A Gold conjugated to 10 nm gold (1:75 in 0.3% BSA and 0.3% CWFS gelatin in PBS, University Medical Center Utrecht) for 60 min, 6 PBS washes over 30 min, 2% glutaraldehyde in PBS for 5 min, 3 water washes for 10 s. After drying, grids were post-stained in 2% uranyl acetate for 4 min, and lead citrate for 1 min.
Images were acquired on TALOS L120 (Thermo Fisher) equipped with a Ceta 4k × 4k CMOS camera. For serial sections, images were aligned in z using the TrakEM2 plugin in FIJI [112 (link)].

Caco-2 cells were grown on 35-mm glass bottom dishes (Matsunami Glass) and prefixed with 4% paraformaldehyde in PBS for 20 min. Then, the cells were stained with spike protein (5 μg/ml) in 1% BSA-PBS for 30 min, followed by incubation with the 20 nm gold particle-conjugated anti-mouse IgG antibody (BBI international, Crumlin; EM. GAT10: 1:100) for 20 min. The samples were fixed again and then stained with an anti-CD133 antibody (18470-1-AP; 1.5 μg/ml), anti-DPP4 antibody (10940-1-AP; 1 μg/ml), anti-Cadherin 17 antibody (CSB-PA006407; 1.5 μg/ml), or anti-VAPA antibody (15275-1-AP; PROTEINTECH; 4.2 μg/ml) for 30 min, followed by incubation with the 10 nm gold particle-conjugated anti-rabbit IgG antibody (BBI international; EM.GAT5: 1:100) for 30 min. The samples were then fixed with 2% glutaraldehyde-PBS at 4 °C overnight, dehydrated using a graded series of ethanol (30%–100%), and embedded in epoxy resin. Next, the samples were cut with an Ultramicrotome (EM UC7, Leica), stained with uranyl acetate, and observed with a transmission electron microscope (Talos L120, Thermo Fisher Scientific).