Ab14781

Briefly, the samples were dehydrated with xylene and alcohol. Antigen retrieval was performed in 10 mmol/L sodium citrate solution (pH 6.0) at 100°C for 16 min, and the samples were cooled for 30 min. Endogenous peroxidase activity using 3% hydrogen peroxide and blocked with foetal bovine serum for 30 min. Thereafter, the slides were incubated with anti-ADCY6 rabbit polyclonal antibodies (ab14781, 1:500, Abcam, USA), anti-N-cadherin (ab76011, 1:100, Abcam, USA), anti-vimentin (ab92547, 1:200, Abcam, USA), anti-Ki-67(ab16667, 1:200,Abcam, USA), anti-E-cadherin(ab40772, 1:500, Abcam, USA) and anti-twist1(ab175430,1:200, Abcam, USA) at 4°C overnight. Then, the samples incubated with biotinylated secondary antibody at 37°C for 30 min. The samples were stained with DAB (3, 3- diaminobenzidine) and Mayer’s haematoxylin.
ADCY6 staining classifications were as followings: range 0–3: 0, negative; 1, weak; 2, moderate; and 3, strong, the percentage of positive cells range 0–4: 0, negative or <5%; 1, 6%–25%; 2, 26%–50%; 3, 51%–75%; and 4, 76%–100%. Used the percentage of positive cells and the intensity to determine the final staining scores. Grades <4 were defined as low ADCY6 expression, while Grades ≥4 were defined as high ADCY6 expression.

Total protein was isolated from breast cancer tumor tissues, paired normal adjacent tissues and cell lines by protein extraction buffer (RIPA lysis buffer). Total protein was separated on 10% SDS–PAGE gels. The membranes for 1 hour at room temperature with 5% nonfat powdered milk. The membranes were probed at 4°C overnight with anti-ADCY6 (ab14781, 1:3000, Abcam, USA), anti-TET1 (ab191698, 1:2000, Abcam, USA), anti-N-cadherin (ab76011, 1:5,000, Abcam, USA), anti-vimentin (ab92547, 1:2,000, Abcam, USA), anti-Ki-67(ab16667, 1:5,000, Abcam, USA), anti-E-cadherin(ab40772, 1:10,000, Abcam, USA), anti-twist1(ab175430, 1:1,000, Abcam, USA) and anti-β-actin (ab8226, 1:5000, Abcam, USA). Then, the membranes incubated with the appropriate secondary antibodies. Finally, protein expression was analysed by chemiluminescence reagents (Hyperfilm ECL,USA).