Mouse anti ck18

Cryostat sections of chimeric mouse livers were stained as previously described^{13 (link)}. Briefly, sections were fixed with acetone and incubated with mouse anti-CK18 (1:400; Dako, Glostrup, Denmark) and human anti-Delta (anti-HDAg-positive human serum, 1:8,000). Specific signals were visualized with Alexa 488- or 546-labeled secondary antibodies (Invitrogen, Darmstadt, Germany). Nuclear staining was achieved by Hoechst 33258 (1:2,000 diluted, Invitrogen, Darmstadt, Germany). Stained sections were then mounted with fluorescent mounting media (Dako, Glostrup, Denmark) and analyzed with the fluorescence microscope BZ9000 (Keyence, Osaka, Japan) using the same settings for the different experimental groups. The percentages of HDAg-positive human hepatocytes were estimated as previously described^{13 (link)} and by using 2 visual fields (displaying approximately 500 human hepatocytes each) from 3 different liver sections per mouse.

Cryostat sections of chimeric mouse livers were stained as previously described (Lutgehetmann et al., 2012 (link)). Briefly, sections were fixed with acetone and incubated with mouse anti-CK18 (1:400, Dako, Glostrup, Denmark), rabbit anti-HBcAg (1:2000, Dako), mouse HLA-ABC (1:50, Antibodies-online, Aachen, Germany), and human anti-Delta (anti-HDAg-positive human serum, 1:8,000). Specific signals were visualized with Alexa 488-, 555-, or 633-labeled secondary antibodies (Invitrogen, Darmstadt, Germany). To enhance the HBcAg staining an anti-rabbit horseradish-peroxidase conjugated secondary antibody (Jackson Immunoresearch, Suffolk, United Kingdom) and the TSA Fluorescein System (Perkin Elmer, Jügesheim, Germany) were used. Nuclear staining was achieved by Hoechst 33258 (1:20,000 diluted, Invitrogen, Waltham, MA, United States). Stained sections were then mounted with fluorescent mounting media (Dako) and analyzed with the fluorescence microscope BZ8710 (Keyence, Osaka, Japan) using the same settings for the different experimental groups. The percentages of HDAg-positive human hepatocytes were estimated as previously described (Lutgehetmann et al., 2012 (link)) and by using 2–5 visual fields (displaying an average of 500 human hepatocytes) per mouse liver.