Ab215199

The HSCR samples were excised from normal and aganglionic segment and then fixed in polyoxymethylene (4%) for at least 48 h. Fixed samples were washed and dehydrated by gradient ethanol, embedded in paraffin and sliced. To analyse the expression location of target DEGs in HSCR samples, IHC for MFAP5 (Abcam, ab203828, Rabbit monoclonal immunoglobulin G (IgG), 1:5000 dilution), THBS4 (Abcam, ab263898, Rabbit monoclonal IgG, 1:600 dilution), POSTN (Abcam, ab215199, Rabbit monoclonal IgG, 1:1000 dilution), ANXA1 (Abcam, ab214486, Rabbit monocle IgG, 1:6000 dilution), HSP70 (Abcam, ab181606, Rabbit monoclonal IgG, 1:1000 dilution), HSP40 (Cell Signaling Technology, 4871T, Rabbit monoclonal IgG, 1:50 dilution), NR2F1 (Abcam, ab181137, Rabbit monoclonal IgG, 1:100 dilution) and ZEB2 (Santa Cruz Biotechnology, sc‐271984, mouse monoclonal IgG, 1:50 dilution) was performed on 4‐μm‐thick paraffin‐embedded formalin‐fixed tissue. The GTVisionTM III Detection System/Mo&Rb kit (GENE, GK500710) was used for IHC detection according to the manufacturer's protocol. Slides were imaged using a Leica DM750 ideal microscope with the Leica ICC50 HD camera.

IHC staining of Ki67 and POSTN was performed according to the manufacturer's protocol. The frozen sections were dried at room temperature, placed in an oven at 37 °C for 10–20 min, fixed with 4% paraformaldehyde for 20 min, and washed thrice with PBS (pH = 7.4) for 5 min. The antigens were then repaired with EDTA (pH 9.0) and endogenous peroxidase was blocked with 3% hydrogen peroxide. The slides were blocked with 3% BSA (G5001‐100 g, Servicebio) at room temperature for 30 min and then incubated with Ki67 (ab16667, Abcam, 1:200) or POSTN (ab215199, Abcam, 1:500) at 4 °C overnight. Finally, the frozen slices were subjected to secondary antibody blocking, DAB staining, nuclear restaining, and dehydration. The protein expression levels of Ki67 and POSTN were evaluated under a microscope by professional pathologists. The expression scores of POSTN in the tumor and stromal regions of 71 FFPE samples were measured according to the positivity percentage (0–5% = 0, 6–25% = 1, 26–50% = 2, 51–75% = 3, >75% = 4) and staining intensity (negative = 0, weak = 1, moderate = 2, strong = 3) (Figure 7A,B). The final score was obtained by multiplying the two scores, which ranged from 0 to 12: Negative = 0, weak = 1–4, moderate = 5–8, strong = 9–12 (Table 1).