Preparation and characterization of a specific antibody for the immunohistochemical detection of L-dopa in paraformaldehyde-fixed rodent brains.

A rat polyclonal antiserum has been obtained after coupling of L-3,4-dihydroxyphenylalanine (L-DOPA) to larger proteins using a low concentration of glutaraldehyde. The antiserum was tested for its affinity and specificity using an enzyme-linked-immunosorbent-assay (ELISA). From competition experiments, the most immunoreactive compound was found to be the non-reduced L-DOPA conjugate. Our specific L-DOPA antiserum enables us to visualize L-DOPA molecule on brain of guinea pigs and rats. We examined the immunohistochemical distribution of the polyclonal L-DOPA antiserum after the fixation of brains with a mixture of paraformaldehyde and picric acid. The presence of L-DOPA-immunoreactive (IR) neurons and fibers was described in the posterior, dorsal and periventricular hypothalamic areas and in the arcuate nucleus. Finally, the distribution of L-DOPA-IR cells was compared to that of tyrosine hydroxylase (TH)-IR cells, by means of a double staining procedure. The presence of two populations of TH-IR cells (TH-positive/L-DOPA-negative and TH-positive/L-DOPA-positive cells) was described in the dorsal part of the hypothalamus.