The localization of tyrosine hydroxylase-like immunoreactivity in the central nervous system: methodological...

The ideal methodologies for the localization of tyrosine hydroxylase-like immunoreactivity were investigated using the quantitative capabilities of the Leitz MPV-3 microspectrofluorometer to determine the best protocol. The following method was found to give the best results. The animals were perfused with the two-stage procedure [1] consisting of an initial perfusion with 4% paraformaldehyde at pH 6.5 and a second perfusion of 4% paraformaldehyde at pH 11. The brains were sectioned on a cryostat and the loose sections were placed in the primary antibody for 80 hours (the time of saturation of the reaction). The primary antibody solution contained 0.1-0.5% lambda-carregeenan and 0.3% Triton-X 100 in phosphate buffered saline. The sections were rinsed and placed in a 0.1-0.3% carregeenan solution with 0.1% Triton-X 100 and containing the secondary antibody. The sections were mounted onto chrome alum subbed slides and allowed to dry. The sections were coverslipped with buffered glycerine (pH 8.6) containing 2 mg/ml paraphenylene diamine as a mounting medium. This medium provided excellent protection from fading but certain subsequent enzyme staining (notably acetylcholinesterase) required the use of buffered glycerine alone. Various counterstains were evaluated for their compatability with the FITC fluorescence. A detailed methodology is presented.