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Methylation-specific PCR was performed using specific primers and conditions previously described (Herman et?al, 1996). Briefly, a 25?��l reaction volume containing 80?ng bisulfite modified DNA, 2��5?��l 10��PCR buffer minus Mg (Invitrogen), 1��5?m?mol/l MgCl2, 0��2?m?mol/l dNTPs, 0��25?��?mol/l specific primer mix and 2 units of Taq DNA polymerase (Invitrogen) were used. Methylation-specific PCR was performed by a thermal cycler (Applied Biosystems, Streetville, Ontario, Canada) under the following cycling conditions: 94��C for 3?min; 40 cycles of 94��C for 45?s, specific annealing temperature for 30?s, 72��C for 1?min 30?s and a final extension of 10?min at 72��C. The sequences of the unmethylation-specific http://www.selleck.cn/products/Bleomycin-sulfate.html primers were 5��-TAGTGTGTAAGTTGTAGGAGAGTGG-3�� (forward) and 5��-CTAAACATAAAAAAATAA CACTAATCCAAA-3�� (reverse), and the sequences of the methylation-specific primers were 5��-GTAGTGCGTAAGTTGTAGGAGAGC-3�� (forward) and 5��-GTAAAAAAATAACGCTAA TCCGAA-3�� (reverse) (Weber et?al, 2005). The sequences of primers used for methylation-specific PCR were localized in the CpG-rich island spanning the coding exon 2 of SOCS3. The annealing temperature for unmethylated and methylated http://www.selleckchem.com/products/MG132.html reactions was 52��C and 54��C, respectively. Immunohistochemical staining was performed using standard techniques. Briefly, formalin-fixed, paraffin-embedded tissue sections of 4?��m thickness were deparaffinized and hydrated. Heat-induced epitope retrieval was performed using citrate buffer (pH 6��0), a pressure cooker and microwave for 20?min. After cooling down, endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 5?min. Tissue sections were incubated with anti-SOCS3 antibody (Santa Cruz Biotechnology) overnight in a humidified chamber at 4��C. They were then reacted with biotinylated MultiLink antibodies (Dako, Glostrup, Denmark) and labelled streptavidin horseradish peroxidase complex http://www.selleckchem.com/products/Rapamycin.html (Dako). The reaction was visualized using 3-3 diaminobenzidene (DAB) chromagen solution (Dako) resulting in brown precipitate. Slides were counter-stained in haematoxylin, rinsed in water and dehydrated with increasing concentrations of ethanol and then xylene. Scoring for SOCS3 expression was assessed independently by two of the authors, and major discrepancies between the two observers (generally