Brian Scott wrote:
>> Hello,
>> I'd like to know if anyone could recommend a long lasting label which would
> allow us to track the migration of newborn cells within the rat CNS and
> allow us to visualize the cells for in vitro recording at their final
> locations. The ideal situation would be to microinject the label into the
> area of interest and wait for three to four weeks. Then visualize the cells
> in vitro with fluorescence and then do patch clamping when the cells are at
> their final locations. Has this been done before? Can a fluorescent probe
> last for this length of time in vivo?
>> Thanks.
>> Brian
Brian,
You might try using some of the carbocyanine dyes on the market.
These are lipophylic dyes that get incorporated into the lipid
bi-layer and will actually show up in processes extending from
growing cells into which it is incorporated. Cell-cell contact does
let it spread somewhat and that may be a problem. They come in
various "flavors" such as Di-I, Di-O etc. which fluoresce at
different wavelengths. In fact there have been double labeling
studies done with these. I believe a company called "Molecular
Probes" markets some of them. For In-vivo injection I have used
DMSO as a solvent and pressure injected the dyes into brain tissue.
These dyes work great in fixed tissue as well. They give you
beautiful color pictures! Another option (somewhat high tech) is
the use of virses to transfect cells with the DNA for "Green
fluorescent protein" which can then be visualized under typical
fluorescent optics. There is also now a red version. There have
been several studies using viruses to ID cells during development.
Some of my collegues and I are now trying to use this technique to
ID infected neurons and by modifying optics used for brain slice
recording direct the experimenter to the infected cell to "poke" and
record. Currently in very early stages so no progress to report.
Good luck with your studies and feel free to drop a line if you have
any questions.
Jerry Clayton
Neurology
Univ. of Colorado Health Sciences Center
Denver, Colorado
USA
