Ap conjugated sheep anti dig antibody

In situ hybridization (ISH) was performed on frozen tumor sections as previously described^{34 (link)}. In brief, 10 μm-thick sections were hybridized with antisense probes to Twist1 and Snai1 overnight at 65°C. After hybridization, sections were washed and incubated with AP-conjugated sheep anti-DIG antibody (1:2000; Roche) for 90 min at room temperature. After three washes, sections were incubated in BM Purple (Roche) until positive staining was seen. Digoxigenin labeled in situ riboprobes were generated by in vitro transcription method (Promega and Roche) using a PCR template. The following primers were used to generate the template PCR product. Twist1; forward (5’-CGGCCAGGTACATCGACTTC-3’) and reverse (5’-TAATACGACTCACTATAGGGAGATTTAAAAGTGTGCCCCACGC-3’) Snai1: forward (5’-CAACCGTGCTTTTGCTGAC-3’) and reverse (5’-TAATACGACTCACTATAGGGAGACCTTTAAAATGTAAACATCTTTCTCC-3’)