Ab227680

Freshly-dissected embryo bodies were observed under a stereomicroscope. Parts of the embryos and embryonic hearts were fixed overnight with 4% paraformaldehyde (PFA) at 4°C and then embedded in paraffin wax or optimal cutting temperature compound (OCT). The paraffin sections were stained with H&E for detection of the cardiac structure. To analyze cell proliferation and detect apoptosis in the cardiac OFT, sections from HO or WT individual embryos at E13.5 were stained with anti-PHH3 (ab32107, Abcam, Cambridge, UK) (45 (link)) and in situ Cell Death Detection Kit, Fluorescein (11684795910, Roche, Mannheim, Germany). For detection of the cNNCs, the OFT-containing sections were incubated with anti-Sox10 primary antibody (ab227680, Abcam, Cambridge, UK), washed with phosphate buffered saline solution with 0.1% Tween 20 (PBST), and then incubated with a secondary antibody [goat antirabbit IgG (H + L) (A27039, Invitrogen, Carlsbad, CA, USA)] in fluorescence immunohistochemical assay. Finally, slides were mounted using DAPI medium (SL1841, Coolaber, Beijing, China) and then three discontinuous sections per embryo subjected to fluorescence microscopy on a Leica microscope (DM6000B, Leica, Germany). Signals were quantified using (ImageJ software), with 10 fields that were randomly selected from three cardiac sections used to quantify positive cell number from the fluorogram.

SXOs and primary tissue samples were fixed in 10% formalin for 1 hour and then washed and resuspended in 70% ethanol. After the alcohol wash, SXOs were transferred to the cap of a 1.5 mL cryotube, resuspended in 0.5% agarose gel, and allowed to solidify overnight at 4°C. The following morning, the agarose-organoid molds were transferred to a cassette, dehydrated in 10% formalin, 70% ethanol three times for 30 minutes, 90% ethanol twice for 30 minutes, 100% ethanol three times for 30 minutes, xylene three times for 20 minutes, and embedded in paraffin. Paraffin blocks were sectioned at 4-um thickness. For hematoxylin and eosin (H&E) staining, slides were deparaffinized in xylene (9 min), 100% ethanol (3 min), 95% ethanol (1 min), and stained with hematoxylin (20 sec). Slides were rinsed in water, soaked in clarifier (40 sec), washed in water, and then bluing agent (20 sec). Slides were then rinsed, stained with eosin (20 sec), followed by serial incubation in 100% ethanol (3 min) and xylene (3 min), and then mounted for microscopic examination. Immunohistochemistry (IHC) staining was performed via the Cell Signaling Technology^® citrate unmasking protocol. Antibodies used included an anti-human Sox10 rabbit antibody (1:100, Abcam ab227680) and an anti-human Gp100 mouse antibody (1:100, Abcam ab732, clone HMB45).

Total protein was extracted from HSCR colon tissues (aganglionic, transition, and dilated segments, n = 3) or cells (n = 3) with a radio-immunoprecipitation assay (RIPA) buffer containing Protease and Phosphatase Inhibitor Cocktail (Halt, Thermo Fisher Scientific, United States). Cell lysates were sonicated, protein concentrations were quantified using the BCA method (P0012S, Beyotime, China), and equivalent protein were resolved by 8% SDS-PAGE (G2061-50T, servicebio, China) in reducing conditions and blotted onto a polyvinylidene fluoride (PVDF) membrane (Roche, Basel, Switzerland). Membranes were blocked using 5% Bovine albumin and incubated with the appropriate diluted antibodies (anti-NRG1, 1:1000, ab191139; anti-RET, 1:1000, ab134100; anti-SOX10, 1:1000, ab227680; Abcam, United Kingdom), (anti-ERBB3, 1:1000, 12708, Anti-Akt, 1:1000, 4,691, anti-pAkt, 1:1000, 4,060, Cell signaling technology, United States), (anti-GAPDH, 1:5000, 60004-1-Ig, HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), 1:5000, SA00001-1, HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), 1:5000, SA00001-2, proteintech, China). The protein expression levels were normalized to those of GAPDH. Results are representative of at least three independent experiments.