Gtvisiontm 3 detection system mo rb kit

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.

RNAlater, the QuantiTect Reverse Transcription Kit and the HotStarTaq^® Master Mix Kit were purchased from Qiagen (Valencia, CA, United States). Trizol reagent was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Rabbit anti-BACE1 and mouse anti-neurofilament (NF) antibodies were all purchased from Abcam (Cambridge, MA, United States). Mouse anti-GFAP, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Waltham, MA, United States). The GTVisionTM III Detection System/Mo & Rb Kit and the DAB Detection Kit were purchased from Gene Tech (Shanghai, China). Isoflurane, a mouse anti-TH antibody and manganese (III) 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis (methochloride) (MnTMPyP) were ordered from Sigma (St. Louis, MO, United States).