Simultaneous observation of fluorescent retrogradely labeled neurons and the immunofluorescently labeled fibers apposing them using Fluoro-Gold and antisera labeled with the blue fluorochrome 7-amino-4-methylcoumarin-3-acetic acid (AMCA).
In this paper we describe a method allowing the simultaneous observation of fluorescent retrogradely labeled neurons and the immunofluorescently labeled fibers which appose them. This technique employs the fluorescent dye Fluoro-Gold to label neurons retrogradely, and 7-amino-4-methylcoumarin-3-acetic-acid (AMCA) conjugated to a secondary antiserum as a label for fluorescence immunohistochemistry. Both Fluoro-Gold and AMCA are excited by near-ultraviolet (UV) light, but under UV excitation Fluoro-Gold appears yellow and AMCA appears blue. Thus the two fluorochromes are both visible under a single condition of illumination and can be readily distinguished by color. This allows quick and accurate determination of whether or not immunofluorescent fibers appose retrogradely labeled neurons.
Wessendorf
M W
MW
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455.
Appel
N M
NM
Elde
R
R
D000818:Animals; D003374:Coumarins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000502:physiology; D007106:Immune Sera / Q000276:immunology*; D009412:Nerve Fibers / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D051381:Rats; D013266:Stilbamidines
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D003374:Coumarins; D005456:Fluorescent Dyes; D007106:Immune Sera; D013266:Stilbamidines; C051029:7-amino-4-methylcoumarin-3-acetic acid