Immunohistochemical Detection of HEV, Tryptase, and 5-HT

Tissue sections were prepared as previously described in this section, were deparaffinating, hydrated, water-bath heated for antigen retrieval and blocked with the addition of 3% hydrogen peroxide for immunohistochemistry. Afterward sections were incubated overnight with primary antibody monoclonal mouse anti-HEV ORF2, tryptase and 5-hydroxytryptamine antibody (1:200 dilution; Beijing Protein Institute, Beijing, China). Immunohistochemical staining was performed following the instructions that were included in the Histostain^TM-Plus kit (ZSGB-BIO, Beijing, China). 3,3′-Diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO, Beijing, China) was applied for 5 min to visualize the antigen–antibody compound, Gill’s hematoxylin was applied as the background stain (Yang et al., 2018 (link)). The slides were observed under light microscope (Olympus Optical Co., Ltd., Beijing, China) and positive signals for HEV-ORF2 proteins were represented by a brown or yellow granular mass (Soomro et al., 2016 (link)). The positive staining intensity of ORF2, tryptase, and 5-hydroxytryptamine were measured as the ratio of the stained area to the total field assessed. Multiple views (three fields per section and five sections per animal) were randomly selected and analyzed.