1% Triton X-100 in TBS, then Brefeldin A chemical structure with TBS alone, and finally with APS (0.1 M Tris [pH 9.0], 0.1 M NaCl, 50 mM MgCl2) at room temperature. The slides were incubated in 100 ��l dye solution (338 ��g/ml nitroblue tetrazolium chloride [NBT], 175 ��g/ml 5-bromo-4-chloro-3-indolyl-phosphate 4-toluidine salt [BCIP], and 450 ��M Levamisole [Vector Labs, Burlingame, CA] in APS) at 37��C in the dark. After sufficient color development, they were washed with deionized water for 1 min and then mounted with aqueous mounting medium. RT-PCR-ISH for detecting HBV RNA. The OCT-embedded frozen sections were placed on glass slides. After proteinase K treatment, the tissue sections were digested with RNase-free DNase I (Roche; diluted to 3 U/��l in 0.1 M sodium acetate and 5 mM MgSO4).
The DNase I reaction mixture (66 ��l) was overlaid onto the tissue sections, which were then enclosed in a frame. The slides were reacted in an aluminum box at 37��C for 20 min and inactivated at 97��C for 10 min. They were then washed in DEPC-treated water, dehydrated in 99.5% ethanol, and air dried. Moloney murine leukemia virus (MMLV) reverse transcriptase (10 U/��l; Invitrogen, Carlsbad, CA) was used in a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5.0 mM MgCl2, 1 ��M antisense primer, 1 mM dNTPs, 2 U/��l RNase inhibitor (Takara, Otsu, Japan), and 10 mM dithiothreitol (DTT). The specimens were then overlaid with the mixture, reacted at 42��C for 60 min, washed with distilled water, dehydrated in 99.5% ethanol, and air dried. The subsequent procedures were the same as described for PCR-ISH.
Immunohistochemical staining for detecting HBV proteins. Deparaffinized formaldehyde-fixed sections or fixed frozen liver tissue sections on glass slides were soaked in distilled water, digested with 0.1% pronase (protease P8038 XXIV; Sigma-Aldrich, Tokyo, Japan) for 1 min, and washed with PBS at room temperature. After 30 min of incubation in blocking reagent (1% BSA and 2.5 mM EDTA in PBS) at room temperature, the slides were reacted with 100 ��l of anti-HBs and anti-HBc polyclonal antibody solutions for 3 h at room temperature and then overnight at 4��C. The following polyclonal antibodies were used: anti-HBs rabbit polyclonal antibody anti-HBc rabbit polyclonal antibody (Novocastra Laboratories, Newcastle, United Kingdom), or normal rabbit serum diluted in blocking reagent.
After the reaction, the slides were washed four times with PBS at room temperature and incubated for 60 min at room temperature in 100 ��l anti-rabbit IgG conjugated with peroxidase (Amersham ECL; GE Healthcare, Piscataway, NJ) diluted to 1:100 in blocking reagent. The slides were then washed four times with PBS at room temperature and stained by using 3,3��-diaminobenzidine tetrahydrochloride (DAB) (Vector Labs). Following counterstaining with Mayer’s hematoxylin solution, AV-951 the tissue specimens were dehydrated in 99.