) and anti-DVL3 (1:1000, BioVision Incorporated.) this website overnight at 4��C. The following day, the blots were washed, and then incubated with horse anti-rabbit secondary antibody (1:2000 dilution; Maixin Biotechnology, Fuzhou, China) for 90 minutes at 37��C and detected by an enhanced chemiluminescence (ECL, Pierce Biotechnology, Rockford, IL, USA). The grayscale values of the DVL-1 and DVL-3 bands were normalized to the values of the corresponding ��-actin band to determine the expression level of the protein. The experiments were repeated 3 times independently. Statistical analysis The Statistical Program for Social Sciences, version 13.0 (SPSS, Chicago, IL), was used for statistical analysis. A t test was used to compare the expression level of DVL-1 and DVL-3 between aganglionic colon segments and ganglionic colon segments.

All results were expressed as means �� standard deviation (S.D.), where P values less than 0.05 were considered statistically significant. Results qRT-PCR analysis The OD value of RNA calculated by A260/A280 was from 1.8 to 2.0. In the course of the qRT-PCR, the amplification curve was received by fluorescent threshold and cycle, a fair reproducibility of each sample and basically coincident efficacy amplification were demonstrable. It was showed that the mRNA levels of DVL-1 and DVL-3 were 2.06 fold and 3.12 fold higher in aganglionic colon segment tissues than those in ganglionic colon segment tissues by the qRT-PCR (n = 50, P < 0.03; n = 50, P < 0.03), respectively (Tables 1 and and22).

Table 1 The relative quantity of DVL-1 mRNA in two segments Table 2 The relative quantity of DVL-3 mRNA in two segments Immunohistochemical staining results The aganglionic and ganglionic colon segments were first defined by the absence of the focal colon ganglion cells through H&E staining (Figure 1). The positive reaction mainly located in the mucous layer and submucous layer of colon segment. The brown yellow depositions in the aganglionic colon segment tissues were far more rich and widespread in submucosa, and were reticulodromous Drug_discovery in the circular muscular layer; while those in the ganglionic colon segment tissues were punctiform. DVL-1 and DVL-3 immunoreactivity showed the tendency of regional increase from ganglionic colon segment to aganglionic colon segment (Figures 2 and and3)3) with significant deviation (Table 3). Besides, immunohistochemical staining was also found that fibrous tissue of hyperplasia between the inner circular and outer longitudinal muscle layer in the aganglionic colon segment could be stained dark yellow by DVL-3, while the plexus wasn��t colored in the ganglionic colon segment (Figure 4). Figure 2 Expression of DVL-1 detected by immunohistochemistry in aganglionic and ganglionic colon segment tissue.