Tissue sections from the gastrointestinal tracts of six dogs were deparaffinized through baths of LMR-SOL (1-bromopropane, 2-methylpropane-2-ol, and acetonitrile), followed by rehydration with successive baths of 100, 90, 70, and 50% ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in PBS. Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in PBS. H and A antigen detection was then performed as previously reported (23 (link)). To assess the ability of VLPs to bind to tissue sections, after blocking, 1 μg/ml VLPs was incubated with the sections overnight at room temperature. Anti-HuNoV primary antibody was then incubated with the tissue sections for 1 h at 37°C. After three washes in PBS, sections were incubated with secondary anti-rabbit biotinylated antibody (Vector Laboratories, Burlingame, CA) diluted in 1% BSA in PBS for 1 h. Sections were washed three times in PBS prior to addition of HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA) also diluted in 1% BSA in PBS. Substrate was added to the slides (AEC kit; Vector Laboratories, Burlingame, CA), followed by Mayer's hematoxylin solution (Merck, Whitehouse Station, NJ) for contrast staining.
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