Immunohistochemical staining of wholemount major pelvic ganglia (MPG) for analysis of myelinated bladder afferents
Peregrine B Osborne, Janet R Keast, Nicole Wiedmann
Abstract
This protocol describes immunohistochemical procedures applied to wholemount major pelvic ganglia (MPG) for the visualization of myelinated axons filled with cholera toxin subunit B (CTB) and/or neurons transfected with adeno-associated virus (AAV) expressing TdTomato. In this protocol, samples were obtained from rats in which CTB was microinjected into the bladder, and AAV-PHP.S was intravenously administered to preferentially and sparsely label peripheral neurons. In this context, neurofascin (paranode marker) identifies myelinated axons.
Steps
Immunohistochemistry
Wash whole MPGs in phosphate buffer (PB; 0.1M; pH 7.2) (3 x 30 min)
Incubate sections in blocking solution (PB; 10% horse serum; and 0.5% Triton-X) at room temperature for 2 h
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.
Wash tissue in PBS (3 x 30 min)
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 24 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.
Wash tissue in PBS (3 x 30 min)
Mount tissue onto glass slides and coverslip in preferred anti-fade mountant.
