(A) The immunofluorescence of PGP9.5 observed by confocal microscopy. The immunopositive areas displayed green fluorescence, and the nuclei showed DAPI’s blue fluorescence. The sham CES group displayed little green fluorescence of PGP9.5, while both control and CES group presented remarkable green fluorescence. (B) The quantitative analysis was performed by ImageJ software, and the results were calculated as follows: area with green fluorescence (%) = (the area stained green by antibody/the area stained blue by DAPI) × 100(%). The data were represented as mean ± S.D. (***P<0.001 versus CES). (C) Representative immunohistochemical staining of PGP9.5. The immunopositive areas displayed brown in the regions of the myenteric plexus, and the nuclei were counterstained blue by Hematoxylin and evenly distributed across the section. There were a large number of PGP9.5 expressing cells in the colonic intramuscular layer in both control and CES groups, and significantly down-regulated PGP9.5-immunopositive cells in the sham CES group. Immunopositive areas were detected by an inverted microscope (Nikon, Japan). (D) ImageJ software was employed for the quantitative evaluation of the immunopositive areas. The relative PGP9.5-immunopositive area (%) (compared with the control group) was used as the y-axis. The data were represented as mean ± S.D. (****P<0.0001 versus CES).