A simple, fast and reliable method for obtaining dopamine histofluorescence from mesencephalic cultures.
A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.
Futamachi
K J
KJ
Veterans Administration Medical Center, Reno, Nevada 89520.
Pisani
P L
PL
Peacock
J H
JH
D000818:Animals; D002478:Cells, Cultured; D004298:Dopamine / Q000032:analysis*; D006038:Glyoxylates; D006651:Histocytochemistry / Q000379:methods*; D008636:Mesencephalon / Q000032:analysis* / Q000166:cytology; D051379:Mice
D006038:Glyoxylates; C031150:glyoxylic acid; D004298:Dopamine