Sc 6153

Tissue samples were obtained from patients with GBC who underwent resection at Kyushu University Hospitals (Fukuoka, Japan) from 2001 to 2012. The approval for the use of tissues was obtained from patients in accordance with the Ethical Committees of Clinical Study at Kyushu University. Paraffin sections of GBC were deparaffinized and rehydrated. The sections were bleached in 3% H₂O₂ for 30 min and then incubated in 10% rabbit serum with primary antibody for Gli1 (SC-6153, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Shh (SC-1194, 1:100, Santa Cruz Biotechnology), or Smo (SC-6366, 1:100; Santa Cruz Biotechnology) at 4°C overnight. The samples were incubated with Histofine Simple Stain MAX PO (G) (Nichirei, Tokyo, Japan) and 3,3′-diaminobenzidine, and visualized using a hematoxylin counterstain. To estimate the number of positive cells, 10 independent areas were selected and total positive cell numbers in that area were counted. If more than 50% of the tumor cells were stained, protein expression was considered to be positive. The cut-off line for determination of positive cells was determined previously.8 (link)

Protein samples were extracted using M-PER reagent (Thermo Fisher Scientific, Chicago, IL, USA). Immunoblotting was carried out with primary antibodies against Gli1 (SC-6153, 1:200), Shh (SC-1194, 1:200), Smo (SC-6366, 1:200), MMP-2 (SC-10736, 1:200), MMP-9 (SC-6840, 1:100), E-cadherin (SC-7870, 1:200), vimentin (SC-5565, 1:500) (all Santa Cruz Biotechnology), or α-tubulin (T6199, 1:1000; Sigma-Aldrich). Blots were developed with the ECL plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ, USA).