Immunohistochemical labelling of lower urinary tract afferents in spinal cord

Cryoprotect fixed tissue (L5-S2 spinal cord) in phosphate-buffered saline (PBS; 0.1 M, pH7.2) containing 30% sucrose. This should be performed at 4C, 24-72h prior to cutting.

Embed tissue in cryomold using OCT, freeze in cryostat and cut sections (40 µm), collecting sections progressively across sets of 4 wells to collect 160 µm spaced series.

Wash sections in PBS (3 x 10 min)

Incubate sections in blocking solution at room temperature for 2 h

Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 48-72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.

Wash sections in PBS (3 x 10 min)

Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 4 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.

Wash sections in PBS (3 x 10 min)

Mount sections onto glass slides and coverslip in preferred anti-fade mountant.

Labeled lower urinary tract afferents with cholera toxin subunit B should be most visible along the boundaries of each dorsal horn. Look for the lateral projections, which are the most prominent, as well as the medial projections, which are comparatively fainter.

Urethra injections of cholera toxin subunit B will occasionally result in the uptake of tracer by preganglionic neurons if the tracer injection site is near intramural or serosal pelvic ganglion neurons. These neurons will be visible within the sacral preganglionic nucleus in the intermediolateral column. If the urethral rhabdosphincter is exposed to tracer, this will be evident by labelling of motor neurons in the ventral horn.