Cell and Neuronal Tracing

Cholera toxin originates from Vibrio cholerae and is comprised of subunits A and B. Subunit A is an ADP-ribosyltransferase that disrupts protein signaling and will lead to cellular dehydration. Alternatively, subunit B is nontoxic and is involved in the binding of ganglioside GM1 to label lipid rafts (Figure 1). In neurons, cholera toxin subunit B binds to glycosphingolipids in axonal membranes. Introduced via microinjection or iontophoresis, cholera toxin subunit B are fixable and photostable retrograde tracers.

While cholera toxin subunit B Alexa Fluor conjugates offer sensitive and selective binding and neuronal tracing, cholera toxin subunit B biotin-XX and horseradish peroxidase conjugates are available for use with tyramide signal amplification (TSA).

Learn more about cholera toxin b and other protein conjugates

DiI and its derivatives are lipophilic tracers, or carbocyanine dyes, that label cell membranes without visibly affecting cell viability. These dyes are weakly fluorescent in water but are highly fluorescent in membranes where they diffuse laterally in the plasma membrane. Lipophilic tracers are useful for live cell staining in tissue sections, in vivo neurons (Figure 2), and cultured cells. Additionally, they can be used in fixed tissue sections, where they can be used to trace neuronal projections (Figure 3). DiI and its derivatives can also be used for long-term studies.

These lipophilic tracers can be loaded via microinjection or iontophoresis and used in both anterograde and retrograde transport studies. They come as crystal and paste preparations which provides convenient options for loading tissues.

Learn more about lipophilic dyes for membrane labeling

Hydrazides and biocytins are fixable and photostable polar tracers. They have low molecular weights (<1,000 Da) and bright fluorescent signals. Hydrazides and biocytins can be used to investigate cell-cell and cell-liposome fusion. These cell-impermeant polar tracers can also be used to trace neuronal projections and investigate gap junctions (Figures 4, 5). Since they can cross gap junctions, biocytins and hydrazides are often used to investigate neuronal communication along with labeled dextrans, which are retained within the cell (Figure 6). Biocytins can also be detected using fluorescent streptavidin conjugates for signal amplification such as TSA and ELF technologies.

These polar tracers can be loaded using microinjection, pinocytosis, or other techniques that temporarily permeabilize the cell membrane. Additionally, electroporation can be used to transport these tracers into cells.

Learn more about fixable polar tracers