Superblock

HuNoV-infected Vero cells were freeze-thawed 2× then the supernatants were transferred to high binding ELISA plates (Corning, Corning, NY, USA) and incubated at 4 °C overnight. The plates were washed 3× with PBS before blocking 2× with SuperBlock (Thermo Fisher Scientific) for 15 min at RT. Following the incubation, a 1:1000 dilution of either a polyclonal rabbit IgG anti-VP1 antibody (Abcam, Cambridge, UK) or a polyclonal rabbit IgG anti-p48 antibody (gift from Christiane Wobus) in SuperBlock was added to the wells for 1 h at 37 °C. After removal of the solution, the wells were washed 3× with KPL wash buffer and a 1:1000 dilution of HRP-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) in SuperBlock was added to the wells and incubated at 37 °C for 1 h. The solution was decanted and the cells were washed 3× with KPL wash buffer and a TMB ELISA Substrate Solution (Thermo Fisher Scientific) was added for colorimetric visualization. OD₄₅₀ was read using an Epoch Microplate Spectrophotometer (Biotek).

Serial 5 μm thick sections from formalin-fixed and paraffin-embedded specimens were deparaffinized and rehydrated through xylene and alcohol. Endogenous peroxidases were blocked in methanol solution with 3% hydrogen peroxide for 20 min; after this, sections were placed in water for 5 min. Subsequently, sections were placed for three times in washing solution TBS1X/0.1% Tween-20 (5 min each time). Then, sections were incubated for 5 min at room temperature with serum (ScyTek Laboratories, Super Block) for blocking non-specific sites, and later with LOX-1 (Abcam, ab60178) and VEGF-A (Santa Cruz, sc-7269) primary antibodies, diluted in TBS1X/BSA 2%. After one hour, sections were washed with TBS1X/0.1% Tween 20 solution as above, and then incubated for 15 min at room temperature with secondary antibody (ScyTek Laboratories, UltraTek Anti-Polyvalent Biotinylated Antibody). After repeating three washes, sections were incubated with Streptaividine solution (ScyTek Laboratories, UltraTek HRP) for 15 min. The staining was completed after a short incubation with a freshly prepared substrate-chromogen, 3-Amino-9-Ethylcarbazole (AEC) (ScyTek Laboratories). The sections were washed extensively in water and nuclei were counterstained with hematoxylin. The intensity of LOX-1 and VEGF-A staining was scored as negative/weak (−), moderate (+), and strong (++).