008 to 1.0 ��g/ml for the frozen sections) in 50 mM Tris (pH 7.5) at 37��C for 30 min in a humidified chamber. Subsequently, proteinase things K was inactivated at 97��C for 10 min, and then the sections were rinsed with distilled water, dehydrated in 99.5% ethanol, and air dried. Primers and probes for PCR-ISH and RT-PCR-ISH. The primers used to amplify the S and X regions of HBV and the 5�� untranslated region (5��-UTR) of HCV as well as the corresponding probes are listed in Table Table2.2. We created a digoxigenin (DIG)-dUTP tail at the 3�� end of the 5��-DIG probe using a DNA tailing kit (Roche, Basel, Switzerland). TABLE 2. Primers and probes for PCR-ISH and RT-PCR PCR-ISH for detecting HBV DNA. PCR was performed by using one of two sets of antisense and sense primers complementary to the sequences located in the S and X regions of HBV.

The PCR mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3.0 mM MgCl2, 0.8 mM each primer, 197 mM deoxynucleoside triphosphates (dNTPs), and 10 U/50 ��l Taq DNA polymerase (AmpliTaq Gold; Applied Biosystems). The tissue slides were warmed to 70��C, and 50 ��l of the PCR mixture was overlaid onto the proteinase K-treated tissue specimens. An Ampli cover disc with Ampli cover clips (Applied Biosystems) was attached to each specimen. The slides were placed in the GeneAmp in situ PCR system 1000 unit at 70��C. PCR was performed at 95��C for 10 min, followed by 35 to 55 cycles at 95��C for 30 s and 60��C for 2 min and a final extension at 72��C for 10 min. Immediately after the PCR, the slides were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 37��C, washed in 2�� SSC (1�� SSC is 0.

15 M NaCl plus 0.015 M sodium citrate) for 2 min, rinsed with distilled water for 2 min, dehydrated in 99.5% ethanol for 1 min, and then air dried. ISH was performed by mixing the DIG-labeled probe (final concentration, 100 ng/ml) with 65 ��l of hybridization buffer (50% deionized formamide, 4�� SSC, 1�� Denhardt’s solution [0.2% bovine serum albumin BSA, 0.2% polyvinyl pyrrolidone, 0.2% Ficoll 400], 100 ��g/ml denatured salmon sperm DNA, 100 ��g/ml yeast RNA, and 1 mM EDTA) and then adding the mixture to each section, heating to 97��C for 10 min, and cooling to 37��C in decrements of 1��C/min (27). Hybridization was carried out overnight at 37��C. Stringency washes were conducted with the following: 2�� SSC twice for 10 min at 37��C, 0.

03�� SSC for 10 min at 50��C, 0.1% Triton X-100 in TBS (0.1 M Tris [pH 7.5], 0.1 M NaCl) for 10 min at room temperature, and TBS for 5 min at room temperature. After incubation in blocking reagent (0.1 M Tris [pH 7.5], 0.1 M NaCl, 10% sheep serum, 3% BSA) at room temperature for Carfilzomib 15 min, the slides were covered with 100 ��l anti-DIG antibody conjugated with alkaline phosphatase (Roche) and diluted at 1:900 with 1% BSA in TBS at 37��C for 60 min.