Automation hematoxylin

The paraffin blocks from liver biopsies were fixed in 10% neutral buffered formalin were processed into 4–5 µm paraffin sections. The pDC cell surface markers BDCA-2 (1:1,000 dilution of an antibody) and ILT7 (1:1,000 dilution of an antibody) were detected by IHC SP method. The tissue slice was dewaxed and incubated in 3% H₂O₂ (VWR International) for 8 min at room temperature to eliminate endogenous peroxidase activity. After blocking with 5% normal goat serum (Thermo Fisher Scientific, Inc.), the slice was incubated with the corresponding primary antibodies against BDCA-2, ILT7 or TLR-9 at 4°C overnight. After washing with PBS, the slice was incubated in biotin-labeled secondary antibody (1:5,000) at 37°C for 30 min. Next, the slice was incubated in horseradish-labeled streptavidin (0.25 mg/ml) at 37°C for 30 min. The sample was then incubated with DAB Plus (cat. no. K3468; Dako) for 4 min at 37°C, followed by incubation with hematoxylin as the counterstain (Automation Hematoxylin; cat. no. S3301; Dako) at 37°C for 5 min. The slice was then washed with water. Finally, the slice was redyed with hematoxylin for 30 sec at 37°C, sealed and observed under a light microscope (BX63 model; Olympus Corporation).