Sc 1488

Enteroids were fixed with 4% paraformaldehyde at the end of differentiation and recovered from the matrix for whole-mount immunofluorescence staining.⁵² Antigen-retrieval was performed using 10 mM sodium citrate pH 6.0 for 30 min at 80°C followed by pre-incubation (2 h) with 10% donkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100 (Sigma-Aldrich). Enteroids were then incubated overnight at 4°C with goat anti-chromogranin A (1:250; sc-1488, Santa Cruz), goat anti-ghrelin (1:250; sc-10368, Santa Cruz), rabbit anti-GLP-1 (1:100; ab108443, Abcam), rabbit anti-motilin (1:50; in-house developed antibody) or rabbit anti-Notch1 (1:100; D1E11, Cell signaling Technology). After washing, enteroids were stained during 2 h with a secondary antibody CY3 donkey anti-goat (1:800; 715-165-150, Jackson ImmunoResearch) or CY5 donkey anti-rabbit (1:800; 711-175-152, Jackson ImmunoResearch), depending upon the host of the primary antibody. Nuclei were stained with DAPI (2.5 μg/ml, ThermoFisher Scientific). For the negative control, the primary antibody was replaced with normal rabbit serum (Jackson ImmunoResearch). Enteroids were mounted back in a 96- CELLSTAR®-plate (Greiner) with Mowiol® 4-88 (Sigma-Aldrich) and visualized using the Operetta CLS High-Content Imaging system (Perkin Elmer). Counting was performed on at least 3-4 views from ± 10 enteroids per patient.

Immunostaining was performed using Vectastain elite ABC peroxidase kit (Vector Laboratories, Burlingame, CA) as described previously³³ . Primary antibodies include HOXB13 and Chromogranin A (CHGA) (sc-28333 and sc-1488 respectively, Santa Cruz Biotechnology, Dallas, TX), FOXA2 (ab 23306-100, Abcam, Cambridge, MA), cytokeratin 5 (CK5) and 14 (CK14) (904,801 and 905,501 respectively, BioLegend, San Diego, CA). The tissue sections were counterstained, mounted, and imaged with a Zeiss microscope (White Plains, NY). The staining was evaluated using a semiquantitative H-score. Immunofluorescence staining was imaged with a Nikon fluorescence microscope (Melville, NY).

IHC staining was performed using Vectastain elite ABC peroxidase kit (Vector Laboratories, Burlingame, CA) as described previously (37 (link)). Primary antibodies include PKM1 and PKM2 (7067S, dilution 1:500, and 4053S, dilution 1:1000, respectively, Cell Signaling Technology, Danvers, MA), P63 and FOXA2 (ab735 and ab108422, respectively, dilution 1:500, Abcam, Cambridge, MA), HOXB13 and chromogranin A (CHGA) (sc-28333 and sc-1488, respectively, dilution 1:200, Santa Cruz Biotechnology, Dallas, TX), FOXA1 (A15278, dilution 1:500, Abclonal, Woburn, MA), Synaptophysin (SYP) (611880, dilution 1:1000, BD biosciences, San Jose, CA), and NKX3.1 (0314, dilution 1:500, Athena Enzyme Systems, Baltimore, MD). Images were taken using a Zeiss microscope (White Plains, NY). The intensity score was evaluated by a semiquantitative blinded manner and graded as 0 (negative), 1⁺ (low), 2⁺ (moderate), and 3⁺ (high). For IF staining, primary antibodies include CHGA (AB_1553436, dilution 1:50, Developmental Studies Hybridoma Bank, Iowa City, IA), PKM1, PKM2, P63, and SYP (source was the same as mentioned above, dilution 1:100). The IF staining was imaged with a Nikon fluorescence microscope (Melville, NY) as reported previously.