Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods.
For ultrastructural studies, it is of great interest to be able to combine anatomical tracer techniques with sensitive immunohistochemical methods. Fluorogold (FG) is a fluorescent and retrogradely transported anatomical tracer, which is commonly used to label neurons in the brain and spinal cord for light microscopic studies. We here describe a method for detecting FG-labeled somata in the electron microscope using a high resolution post-embedding immuno-gold method. For this purpose, spinal motoneurons were retrogradely labeled by an intraperitoneal injection of FG in the adult rat. The rats were intravascularly perfused with a fixative solution containing 2% paraformaldehyde and 1-2% glutaraldehyde. Vibratome sections of spinal cord tissues were cryo-protected in glycerol, freeze substituted in methanol containing uranyl acetate, and embedded in the Lowicryl HM20 resin at low temperatures. Electron microscopic analysis demonstrated atypical lysosome-like structures in the cytoplasm of FG-labeled motoneurons. Subsequent post-embedding immuno-gold labeling demonstrated prominent accumulation of FG in numerous lysosomes but not in other organelles or cytoplasmic compartments of the labeled neurons. The protocol is versatile and allows for combining anatomical tracing of neurons with, e.g., neuro-transmitter studies in the electron microscope. We suggest that the described method for sensitive detection of FG in the spinal cord may also have broad applicability to other areas of the central nervous system.
Persson
Stefan
S
Department of Neurology, David Geffen School of Medicine at UCLA, Neuroscience Research Building, 635 Charles E. Young Drive South, Los Angeles, CA 90095, United States.
Havton
Leif A
LA
D000818:Animals; D003593:Cytoplasm / Q000187:drug effects / Q000648:ultrastructure; D005260:Female; D007150:Immunohistochemistry / Q000379:methods*; D008247:Lysosomes / Q000187:drug effects / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009046:Motor Neurons / Q000187:drug effects / Q000648:ultrastructure*; D009457:Neuroglia / Q000648:ultrastructure; D056325:Neuronal Tract-Tracers / Q000493:pharmacokinetics*; D015388:Organelles / Q000187:drug effects / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000187:drug effects / Q000648:ultrastructure; D013266:Stilbamidines / Q000493:pharmacokinetics*
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D056325:Neuronal Tract-Tracers; D013266:Stilbamidines