Anti hbcag primary antibody

Liver tissue samples were fixed in 4% buffered formalin for 48 h and were paraffin embedded. Liver sections that were2-μm-thin were then prepared with a rotary microtome (HM355S, ThermoFisher Scientific, Waltham, USA). Immunohistochemistry was performed using a Bond Max system (Leica, Wetzlar, Germany, all reagents from Leica) with the anti-HBcAg primary antibody (Diagnostic Biosystems, Pleasanton, CA; 1:50 dilution) and a horseradish peroxide coupled secondary antibody. Briefly, the slides were deparaffinized using deparaffinization solution pre-treated with epitope retrieval solution (corresponding to citrate buffer pH6) for 20 min. Antibody binding was detected with a polymer refine detection kit without post primary reagent and was visualized with DAB as a dark brown precipitate. Counterstaining was done with haematoxylin. Slides were scanned using a SCN 400 slide scanner (Leica Biosystems, Nussloch, Germany), and HBcAg-positive hepatocytes were determined based on the localization, intensity, and distribution of the signal in 10 random view fields (40× magnification). The mean numbers of the HBcAg-positive hepatocytes were quantified per mm2.

Liver tissue samples were fixed in 4% buffered formalin for 48 h and embedded in paraffin. Subsequently, 2 µm thin liver sections were prepared using a rotary microtome (HM355S, ThermoFisher Scientific, Waltham, MA, USA). Nuclei were stained with hematoxylin. The following antibodies were used to stain HBcAg, CD3 and CD3+Ki67: anti-HBcAg primary antibody (Diagnostic Biosystems, Pleasanton, CA; 1:50 dilution) and a horseradish peroxide coupled secondary antibody; anti-CD3 antibody (host: rabbit (IR503; Dako); ready to use) and anti-Ki67 antibody in a 1:200 dilution (SP6, NeoMarkers/Lab Vision Corporation). For quantification, sections were scanned and scored manually (40× magnification): in 6–10 randomly chosen areas of 0.5 mm² on CD3-stained paraffin sections, cells (CD3+ and HBc+) were counted and merged for statistical analysis. For counts of T cell clusters, cell accumulations of >3 CD3+ cells and >6 CD3+ cells were counted in the whole sections and normalized to cell accumulations/mm².