Anti panck

Formalin-fixed paraffin-embedded CRC-LM tissue sections were deparaffinized in xylene, and rehydrated in a series of methanol dilutions (see above). Antigen retrieval was done with the AR6 buffer (Perkin Elmer, Waltham, MA, USA; cat. no.: AR600250), and then sections were incubated in serum-free blocking solution for 30 min (Agilent-Dako, Santa Clara, CA, USA; cat. no.: X0909). Sections were then incubated with the primary antibody at 4 °C overnight, washed in PBS, and incubated at RT with the secondary antibody Histofine MAX PO Multi (Nichirei, Tokyo, Japan; cat. no. 414152F) for mouse and rabbit antibodies and Histofine MAX PO G (Nichirei Bio, cat. no. 414162F) for antibodies of goat origin, for 30 min. The following primary antibodies were used: anti-Ki67 (cat. no.: M724029-2, Dako), anti-CD31 (cat. no.: IR61061-2, Dako), anti-Pan-CK (cat. no.: GA05361-2, Dako), anti-β-catenin (cat. no.: M353901-2, Dako), a-smooth muscle actin (cat. no.: GA61161-2, Dako), anti-CADH1 (cat. no.: GA05961-2, Dako), and LTBP2 (cat. no.: AF3850, R&D Systems, Minneapolis, MN, USA). Subsequently, sections were washed in PBS (3 times for 5 min), and stained using the Opal system (Perkin Elmer, cat. no.: NEL810001KT). For further details, see the Supplemental Material section.