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Faculty of Medicine, Dentistry and Health Sciences
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Whitington Lab ~ MethodsHow we stain neurons in the embryoNeurons are transparent and must be stained to visualise their morphology. We use a variety of methods to do this... Method 1 - Antibody staining
One way we stain neurons is by using antibodies that bind to specific proteins only present in neurons. An example is the monoclonal antibody 22C10, which recognises a protein called Futsch found in all sensory neurons and their axons. The bound antibody is visualised by an enzymatic reaction, in which the substance diaminobenzidine (DAB) is converted from a colourless to a blue product by an oxidation reaction. The movie above shows different planes of focus of three adjacent hemisegments in an embryo stained with 22C10. Movie by Veronica Martin. Method 2 - Dye injection
Dye injection: To stain a single, selected neuron we use the method of dye injection. A fine glass micropipette containing a fluorescent dye such as DiI is placed on the surface of a particular neuron and the dye is ejected from the pipette. The fluorescence is then converted into a DAB reaction product by exposing the neuron to fluorescent light. An example of a single lch5 sensory neuron stained with DiI is shown above. Image by Kerri-Lee Harris. Method 3 - GFP expression
Neurons can also be stained by making them express (produce) the non-toxic, fluorescent dye Green Fluorescent Protein. We drive expression of GFP in specific neurons using the GAL4-UAS system (see box below). In the image above, GFP has been expressed in a the axons of a subset of central neurons, including the motor neuron aCC (arrow). In this case, we also stained the RP2 neuron with DiI, enabling us to look at the interaction between the RP2 and aCC axons. Image by Michael Murray. See also related movie.
How we drive gene expression in specific tissues of the embryoWe need to drive expression of a particular gene (X) in a specific cell or tissue of the embryo for a number of reasons, including:
For this purpose, we use the GAL4-UAS system. A line of flies which expresses GAL4 (a transcriptional activator protein) in the tissue of interest (e.g. all sensory neurons) is crossed to another line carrying the UAS element upstream of gene X. In the progeny of this cross, GAL4 in the tissue of interest will bind to the UAS element and activate transcription of the downstream gene X, specifically in that tissue.
from Brand and Perrimon, Development 118, 401-415 (1993) |
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The Whitington Lab. |
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Date Created: 14 Feb 2005 |
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