Neuronal tracing can occur through retrograde (labeling begins at the axon termini and the dye is transported back through the axon to the cell bodies) or anterograde (labeling begins at the dendrites and cell bodies and the dye is transported out to the axons and their terminals) processes. In some cases, tracers work both ways simultaneously, whereas in other cases, the tracers only work one way or the other. Retrograde and anterograde tracing of neurons has been performed using a wide variety of fluorescent and nonfluorescent probes.1 Retrograde and anterograde tracers include lipophilic tracers such as DiI, biotin derivatives, and polar fluorescent dyes (all discussed previously in this section) as well as protein conjugates (including lectins), dextran conjugates, and fluorescent microspheres, described below.
Fluorescent Cholera Toxin Conjugates
Cholera toxin subunit B (CT-B) is an important tool for the retrograde labeling of neurons 2,3 useful for tracing forebrain afferents 4 and projections of the parabrachial region.5 Our Alexa Fluor CT-B conjugates are prepared from recombinant cholera toxin subunit B, which is completely free of the toxic subunit A, thus eliminating any concern regarding toxicity or ADP-ribosylating activity of the molecule. Molecular Probes cholera toxin conjugates are versatile tracers with superior brightness for sensitive and selective receptor labeling and neuronal tracing.
Fluorescent Lectin Conjugates
Phaseolus vulgaris leucoagglutinin (PHA-L) is a widely used lectin for anterograde axonal tracing. Iontophoretically injected PHA-L clearly shows the morphological features of the filled neurons at the injection site and stains the axons and axon terminals. Furthermore, PHA-L that has been transported intracellularly is not degraded over long periods. PHA-L has been used in combination with our biotin dextran amines for two-color experiments.6,7 For retrograde tracing, choose one of our fluorescent wheat germ agglutinin (WGA) conjugates.
NeuroTrace Fluorescent Nissl Stains
The Nissl substance, described by Franz Nissl more than 100 years ago, is unique to neuronal cells.11 Composed of an extraordinary amount of rough endoplasmic reticulum, the Nissl substance reflects the unusually high protein synthesis capacity of neuronal cells. Various fluorescent or chromophoric "Nissl stains" have been used for this counterstaining, including acridine orange, ethidium bromide, cresyl violet, methylene blue, safranin-O, and toluidine blue-O. We have developed five fluorescent Nissl stains that not only provide a wide spectrum of fluorescent colors for staining neurons, but also are far more sensitive than the conventional dyes.
Fluorescent and Biotinylated Dextrans
Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility, and low toxicity. Fluorescent and biotinylated dextrans are routinely employed to trace neuronal projections and can function efficiently as anterograde or retrograde tracers, depending on the study method and tissue type used. Our fixable dextrans in the 3,000 to 10,000 molecular weight range are generally preferred for neuronal tracing because they transport more effectively and can be fixed with aldehydes after labeling. For more choices of dextran conjugates, see Fluorescent and Biotinylated Dextrans for Tracing Applications in this section.
Molecular Probes also offers the NeuroTrace BDA-10,000 Neuronal Tracer Kit. It includes biotin dextran amine (BDA-10,000), HRP-avidin, and DAB. BDA-10,000 is injected into neuronal tissue; the tissue is then fixed, sectioned, and incubated with avidin-HRP. The sample is then treated with DAB, and the electron-dense DAB reaction product can be viewed with an electron or light microscope.