Ab155279

Luciferase assay was performed as described previously^{11 (link),16 (link)}. Dewaxed paraffin sections were used for immunofluorescent staining. The sections were incubated with rabbit polyclonal antibody raised against PITX1 (ab70273, 1:2000; Abcam), SOX9 (ab185966, 1:500; Abcam), SOX10 (ab155279, 1:10,000; Abcam), Ki-67 (pre-diluted, Nichirei, Tokyo, Japan) or cleaved Caspase3 (#9664, 1:400; Cell Signaling Technology) at 4 °C overnight. After being washed in T-TBS, the sections were incubated with secondary Alexa488-conjugated anti-rabbit IgG antibody (#8890, 1:500, Cell Signaling Technology) for 1 h at room temperature. After further washing, coverslips were placed on the glass slides using a water-soluble mounting medium. The slides were observed using fluorescence microscopy. Images were captured using an AxioImagerZ2 fluorescence microscope (Carl Zeiss GmbH, Jena, Germany) with a 40 × objective.

Western blotting was performed as described previously^{37 (link)}. Membranes were blotted with rabbit polyclonal antibody against human PITX1 antigen (ab70273, 1:2,000; Abcam, Cambridge, MA, UK), rabbit monoclonal antibody against human SOX9 antigen (ab185966, 1:2000; Abcam), rabbit monoclonal antibody against human SOX10 antigen (ab155279, 1:2000; Abcam), mouse monoclonal antibody against FLAG antigen (F1804, 1:2000; Sigma), or with polyclonal antibody against α-tubulin (PM054-7, 1:5000; MBL, Tokyo, Japan) and the appropriate standard peroxidase-labeled anti-mouse IgG and anti-rabbit IgG secondary antibodies, according to the manufacturer's instructions (GE Healthcare, Piscataway, NJ, USA). Immunoreactive bands were visualized using the ECL detection system (Pierce, Rockford, IL, USA).

Immunohistochemistry was performed on recipient mouse colon, as previously described [7 (link), 22 (link)]. Wholemount preparations of the LMMP and enteric neurospheres were fixed in 4% paraformaldehyde. Wholemount LMMP or neurosphere preparations were permeabilized with 0.1% Triton X-100 and blocked with 10% donkey serum. Primary antibodies were diluted in 10% donkey serum and included goat anti-GFAP (1:200, Abcam, ab53554), human anti-HuC/D (Anna1, 1:16,000, kindly gifted by Lennon laboratory) mouse anti-HuC/D (1:50, Invitrogen, A-21271), rabbit anti-nNOS (1:100, Cell Signaling, C7D7), rabbit anti-Sox10 (1:400, Abcam, ab155279), and mouse anti-neuronal class III conjugated β-tubulin (Tuj1; 1:400; Covance, Dedham, USA). Secondary antibodies included anti-rabbit IgG (1:500; Alexa Fluor 488; Fisher Scientific Life Technologies) and anti-human IgG (1:200, Alexa Fluor 594; Fisher Scientific Life Technologies). Cell nuclei were stained with DAPI (Vector Labs, Burlingame, CA) and mounted with aqua-poly/mount (Fisher Scientific Polysciences Inc.). Images were taken using Nikon A1R laser scanning confocal microscope (Nikon Instruments, Melville, NY) or Keyence BZX-700 All-In-One Microscopy system (Keyence America, Itasca, IL).

Immunostaining against neural crest stem cell marker (nestin), neural crest cells marker (SOX10), immature neurons markers (doublecortin and β‐III tubulin), and glial marker (GFAP) were performed to verify expanded EPI‐NCSCs. Briefly, cultured EPI‐NCSCs were fixed with 4% paraformaldehyde and washed with PBS containing 0.05% Tween‐20. Then, cells were blocked with 1% bovine serum albumin containing 0.2% triton X‐100 and incubated overnight at 4°C with primary antibodies: rabbit anti‐nestin, anti‐SOX10, anti‐doublecortin, anti‐β‐III tubulin and anti‐GFAP (#ab93157, ab155279, ab77450, ab18207, and ab7260; Abcam). Following washing with PBS, cells were incubated with FITC‐conjugated secondary antibody (Sigma, #F1262) and counterstained with DAPI (Sigma, #D9564). Images were captured with the Olympus inverted fluorescence microscope.

ChIP was performed as described in [9 (link)] using approximately 1x10⁷ SK-MEL-28 clonal CRISPRi cells per assay and 5 μg of the following antibodies: anti-SOX10 rabbit monoclonal antibody (Abcam; ab155279), anti-Histone H3K27ac rabbit polyclonal (Active Motif; #39133) or anti-rabbit IgG control (Merck; PP64). qPCR primers used to amplify SOX10 bound genomic sequences are shown in S7 Table.

A group of sections were randomly selected from the three additional groups of sections from each mouse. The sections were rinsed with PBS-T, successively infiltrated with 2 mol/L HCl for 10 min (only required for the anti-BrdU antibody), subjected to antigen retrieval in citrate buffer (0.01 M, 99°C) for 30 min, and incubated with normal goat serum for 2 h at 37°C. Then, the primary anti-CNPase (ab6319, Abcam), anti-Olig2 (rabbit, ab109186, Abcam), anti-SOX10 (ab155279, Abcam), anti-bromodeoxyuridine (BrdU; ab6326, Abcam), anti-5-HT1AR (ab85615, Abcam), anti-platelet-derived growth factor alpha receptor (PDGFαR; ab96569, Abcam), anti-Aβ (ab11132, Abcam), and anti-CDKN2A/p16INK4a (p16; ab201980, Abcam) antibodies were added at a dilution of 1:500 in PBS, incubated at 4°C for 72 h and then rewarmed at 37°C for 2 h. The appropriate DyLight 405, DyLight 488, and DyLight 549-conjugated secondary antibodies (A23140, A23210, and A23320, respectively, Abbkine, P. R. China) were incubated with the sections at a 1:200 dilution. Finally, the sections were mounted on gelatin-coated slides with antifade solution to reduce fluorescence quenching.