Clone 14

When tissue was available for tumors in which a CTNNB1 mutation was detected, immunohistochemistry to detect localization of β-catenin protein was performed using formalin-fixed, paraffin-embedded sections as previously detailed (clone 14, dilution 1:500; BD Biosciences, San Jose, CA) (13 (link)). When possible, the same paraffin block/mirror image block that was used for sequencing was also used for immunohistochemistry. Presence or absence of nuclear staining was evaluated and percentage of tumor demonstrating nuclear staining was recorded. Presence of membrane staining in other epithelial cells served as an internal positive control. Immunohistochemistry assessment was performed by a trained gynecologic pathologist (RRB).

Fluorescent images were acquired using a Nikon A1 Laser Scanning Confocal microscope. SW480 cells were analyzed for nuclear translocation of β-catenin following Wnt stimulation as described previously (27 (link)). Samples were co-stained with antibodies against total β-catenin (Clone 14, BD Biosciences) and non-phospho serine 45-β-catenin (D2U8Y, Cell Signaling 19807) before counterstaining with Alexa-Fluor 488 and 647, respectively. For quantification, the region of interest generator in Nikon Elements was used to automatically select nuclei positive for DAPI staining. Staining intensity was then exported for each channel of interest on a minimum of 100 cells per treatment. Hyperspectral imaging coupled with linear unmixing was performed to determine the localization of PDE10 in colon tissue. Spectral imaging and analysis was used to separate the autofluorescence signal from the AlexaFluor-488 labelling of PDE10, as described previously (30 (link),31 (link)). A library containing pure spectra of AlexaFluor-488 (PDE10), AlexaFluor-647 (β-catenin), and DAPI (nuclei) was constructed using single labeled controls of HT-29 cells labelled with only AlexaFluor-488, AlexaFluor-647, and DAPI, respectively. Normal colon tissue section was used to obtain the autofluorescence spectral signature. Images were scaled similarly and false colored for visualization as indicated in the text.