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.
Laboratoire de Neurocytochimie fonctionnelle, URA C.N.R.S. Université de Bordeaux I, Talence France.
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D000818:Animals; D000906:Antibodies; D001921:Brain / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D005557:Formaldehyde; D006168:Guinea Pigs; D006652:Histological Techniques; D007031:Hypothalamus / Q000166:cytology*; D007124:Immunoenzyme Techniques; D007980:Levodopa / Q000032:analysis* / Q000276:immunology; D009412:Nerve Fibers / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology; D009928:Organ Specificity; D011108:Polymers; D051381:Rats; D011919:Rats, Inbred Strains; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis
D000906:Antibodies; D011108:Polymers; D005557:Formaldehyde; D007980:Levodopa; D014446:Tyrosine 3-Monooxygenase; C003043:paraform