Proteinase k antigen retrieval solution

Liver tissues were collected and fixed in 4% paraformaldehyde (Sinopharm Chemical Reagent) and embedded in paraffin wax. 5-μm tissue sections were cut, dewaxed and rehydrated through xylene and alcohols and were subsequently subjected to hematoxylin and eosin (H&E) staining for histopathological assessment as previously described^{26 (link)}. For Immunohistochemical (IHC) staining, tissue sections were dewaxed, rehydrated and antigen retrieved using proteinase K antigen retrieval solution (Abcam). Following this, tissue sections were washed three times with Tris-buffered saline (TBS) buffer and incubated with goat anti-Rat IgG (OriGene) as blocking reagent for 15 min. Intrahepatic expression of core antigen of HBV (HBc) was detected by incubating with anti-HBcAg monoclonal Ab (Gene Tech; GB058629) overnight at 4 °C. After washing with TBS buffer, tissue sections were incubated with biotinylated anti-rabbit IgG and streptavidin/horseradish peroxidase conjugates (ZSGB-Bio) for 20 min at 37 °C. DAB substrate was then added followed by counterstaining with hematoxylin. Finally, sections were dehydrated, cleared and mounted for analysis. Images were captured using Olympus BX46 microscope (Olympus, Tokyo, Japan).

Mouse livers, spleens or brains, or whole mosquitoes, were fixed with 10% neutral buffered formalin and embedded in paraffin blocks. Thin sections were then stained with hematoxylin-eosin staining (H.E.) at the Anatomic Pathology Laboratory at UTMB. For IHC, sectioned tissues were treated with proteinase K antigen retrieval solution (Abcam, Waltham MA), followed by blocking with Animal-Free Blocker (Vector Laboratories), anti-RVFV N rabbit polyclonal antibody^{26 (link)}, and biotinylated secondary goat anti-rabbit IgG antibody (Vector Laboratories). Sections were further incubated with streptavidin alkaline phosphatase (Vector Laboratories) and then the ImmPACT Vector Red substrate (Vector Laboratories), before being additionally stained with hematoxylin to visualize the background. Images were captured via cellSens software using a DP74 camera attached to an Olympus IX73 microscope. Positive IHC signals were analyzed under brightfield (magenta) or TRITC fluorescent filter (red) according to manufacturer’s instruction. Merged images of brightfield (reduced brightness) and TRITC fluorescent filter were made by cellSens software.