Immunohistochemistry : 5-HT4R and 5-HT Double Labeling
Biopsies were taken in the proximal, transverse, descending colon and rectum and were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) for 24 hours at 40C and processed for paraffin embedding. Paraffin sections of 4µm were cut, mounted on coated slides, de-waxed and re-hydrated. Antigen retrieval was performed on paraffin sections prior to immunostaining by heating the slides in 0.01M citrate buffer (pH 6.0) for 20 minutes. Non-specific binding was blocked in 5% normal goat serum (NGS, Sigma, St. Louis, MO) for one hour at room temperature. Sections were then incubated in primary antibodies overnight at room temperature and secondary antibodies for one hour at room temperature. Primary antibodies were polyclonal rabbit anti-5-HT4 receptor (Atlas Antibodies, Cat # HPA040591, Lot # R36821, 1:200) and monoclonal mouse anti-5-HT (ThermoFisher Scientific, Cat # MA5-12111, 1:100); secondary antibodies were Alexa 488 goat anti-rabbit IgG (Jackson ImmunoResearch, 1:300) for 5-HT4R staining and Cy3 goat anti-mouse IgG (Jackson ImmunoResearch, 1:200) for 5-HT staining. After rinsing several times, sections were mounted with fluoroshield mounting medium with DAPI (Abcam, Cat # ab104139) for nuclear counterstain. All antibodies were diluted in 0.05M PBS (pH 7.4) with 0.03% triton-X-100. Negative controls included the omission of primary antibodies from the incubation solution.