A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or neurobiotin-labeled neurons.
Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin-peroxidase conjugate, is both faster and less labor intensive than photo-oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti-serotonin as an example.
Sun
X J
XJ
Department of Psychology, Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada.
Tolbert
L P
LP
Hildebrand
J G
JG
Meinertzhagen
I A
IA
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives*; D019921:Biotinylation; D004396:Coloring Agents; D005456:Fluorescent Dyes; D006793:Houseflies; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods; D008239:Lysine / Q000031:analogs & derivatives*; D018402:Manduca; D018613:Microscopy, Confocal / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D009474:Neurons / Q000737:chemistry* / Q000166:cytology* / Q000648:ultrastructure
D004396:Coloring Agents; D005456:Fluorescent Dyes; C070614:neurobiotin; D001710:Biotin; C013411:biocytin; D008239:Lysine