For the immunofluorescence staining of the distal colon and dorsal root ganglions (DRG, from right S2 to S4), deparaffinized and hydrated sections were incubated with normal serum to block the non-specific reaction and then added with a primary antibody of tryptase (1:100; ab2378, Abcam, Cambridge, UK), PGP9.5 (1:100; ab109261, Abcam, Cambridge, UK), TrkA (1:100; cat 06–574, Sigma-Aldrich, Saint Louis, MO), TRPV1 (1:100; ab6166, Abcam, Cambridge, UK) overnight for 4°C. Secondary antibodies were incubated for 1 h at room temperature and DAPI for 5 min. The sections were sealed with anti-fade mounting medium and then observed under an Olympus fluorescence microscope.
Quantifications of tryptase, TrkA and TRPV1 were performed by the Image J software (National Institutes of Health, Bethesda, MD). Immunopositive areas of tryptase or TrkA in the distal colon and TRPV1 in the S2-S4 DRGs, and double-labeled neurons (TRPV1 and PGP9.5) in the distal colon were measured to compare the difference among the groups. Each slide contained 2 to 4 nonconsecutive sections and random microfields were taken from each slide. The average value per each animal was used for statistical analysis.
Quantifications of tryptase, TrkA and TRPV1 were performed by the Image J software (National Institutes of Health, Bethesda, MD). Immunopositive areas of tryptase or TrkA in the distal colon and TRPV1 in the S2-S4 DRGs, and double-labeled neurons (TRPV1 and PGP9.5) in the distal colon were measured to compare the difference among the groups. Each slide contained 2 to 4 nonconsecutive sections and random microfields were taken from each slide. The average value per each animal was used for statistical analysis.