Catalog s1699

A standard immunohistochemistry analysis was performed for the cytokeratin 19 (CK19) and Epithelial cell adhesion molecule (EpCAM) as follows. Formalin-fixed tissue sections were sectioned and placed on positively charged slides and pretreated with steam heating in DAKO target retrieval solution (Catalog #S1699, Agilent, Santa Clara, CA) for 20 min. Slides were incubated with anti-CK19 antibody (Catalog #13092, Cell Signaling, Danvers, MA, dilution 1:100) and anti-EpCAM antibody (Catalog #14452, Cell Signaling, dilution 1:100), overnight stained using the ABC kit (Vector Laboratories, Burlingame, CA) following the manufacturer’s protocols, developed in diaminobenzidine DAB (Invitrogen, Carlsbad, CA), and counterstained with hematoxylin. Assessment of immunohistochemical staining was performed on scanned colon mucosa sections captured at 10 × magnification. H scores were calculated for CK19 or EpCAM positivity in the cancerous regions using QuPath v0.3.0-m4 image analysis software (https://qupath.github.io/).

A standard immunohistochemistry analysis was performed for the phosphorylated S6 ribosomal protein (p-S6), a downstream molecule of the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signal pathway. Ethanol-fixed tissue sections were sectioned and placed on positively charged slides and pretreated with steam heating in DAKO target retrieval solution (Catalog #S1699, Agilent, Santa Clara, CA, USA) for 20 min. Slides were incubated with anti-p-S6 (Ser 235/236) antibody (Catalog #2211, Cell Signaling, Danvers, MA, USA, dilution 1:100), overnight stained using the ABC kit (Vector Laboratories, Burlingame, CA, USA) following the manufacturer’s protocols, developed in diaminobenzidine DAB (Invitrogen), and counterstained with hematoxylin. Assessment of immunohistochemical staining was performed on scanned colon mucosa sections captured at 10x magnification. H scores were calculated for p-S6 positivity in the epithelial and stromal regions of the distal colon using QuPath v0.2.0-m4 image analysis software (https://qupath.github.io/ access date: 1 February 2021).