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.