Dear Steven,
Biologically active peptides labeled with fluorophores (Fluo-peptides)
have been used successfully by Beaudet et al. to visualize in vivo
retrograde transport in neuronal cells using confocal microscopy
(Biochemical Pharmacology 47:43-52, 1994). If NPY neurons possess
autoreceptors, a biologically active fluorescent NPY analog (Fluo-NPY)
is available from Advanced Bioconcept and may be used to study
antero/retrograde transport. Ten additional biologically active
Fluo-peptides are also available and include analogs of deltorphin,
dermorphin, galanin, neurokinin A, neurotensin, PACAP, PYY,
somatostatin, substance P and VIP.
Fluo-peptides can be visualized with very high resolution using either a
confocal microscope or a regular fluorescence microscope. Unlike
radiolabeled peptides, Fluo-peptides permit double labeling of receptor
positive elements with other antigens. You will find that the relative
simplicity of performing and visualizing double labeling using a
Fluo-peptide and a fluorescent antibody compares quite well with the
difficult and often messy process of double labeling with radiolabeled
peptides followed by immunocytochemistry.
The references below include receptor tracing and immuno identification
of neurons, and show examples of the striking photographs generated.
For the use of Fluo-peptides in double labeling & CLSM:
1. Beaudet A, Mazella J, Nouel D, Chabry J, Castel MN, Laduron P,
Kitabgi P, Faure MP. Internalization and intra-cellular mobilization of
neurotensin in neuronal cells. Biochemical Pharmacology 47:43-52, 1994.
2. Gaudriault G, Nouel D, Dal Farra C, Beaudet A, Vincent JP.
Receptor-induced internalization of selective peptidic µ and d opioid
ligands. The Journal of Biological Chemistry (In press).
3. Faure MP, Alonso A, Nouel D, Gaudriault G, Dennis M, Vincent JP,
Beaudet A. Somatodendritic internalization and perinuclear targeting of
neurotensin in the mammalian brain. The Journal of Neuroscience
15:4140-4147, 1995.
For examples of Fluo-peptides in CLSM:
4. Nouel D, Gaudriault G, Houle M, Reisine T, Vincent JP, Mazella J,
Beaudet A. Differential internalization of somatostatin in COS-7 cells
transfected with SST1 and SST2 receptor types: a confocal microscopic
study using novel fluorescent somatostatin derivatives. Endocrinology
138(1):296-306, 1997.
5. Faure MP, Gaudreau P, Shaw I, Cashman NR, Beaudet A. Synthesis of a
biologically active fluorescent probe for labeling neurotensin
receptors. The Journal of Histochemistry and Cytochemistry 42:755,
1994.
Fluo-peptides are used not only for receptor imaging, but for
electrophysiological studies, flow cytometry, cell sorting and drug
screening.
Regards,
Alex Costa
Advanced Bioconcept
1440 Ste-Catherine W., Suite 424
Montreal, PQ H3G 1R8, Canada
514 874-5434 tel
1-888-FLUOTAG
514 874-9077 fax
[log in to unmask]
http://www.bioconcept.com
Dr Steven Thomas wrote:
>
> Dear confocalists,
>
> We are currently interested in doing some studies to look at neurons
> (probably NPY neurons) in using anterograde and retrograde transport of
> the labels. We also want to combine this with immunohistochemical
> identification of the neuronal types (ie dual labelling).
>
> Any reccomendations of references regarding the combination of dye tracing and immuno
> identification of the neurons using confocal laser scanning
> microscopy would be gratefully appreciated.
>
> Thanks
> Steven
>
> In theory, there is no difference between theory and practice.
> In practice, there is a big difference.
> __________________________________________________________________
> Steven Thomas PhD
> Prince Henry's Institute of Medical Research
> Level 4, Block E
> Monash Medical Centre
> Clayton, Victoria 3168.
> AUSTRALIA
>
> Tel: +61 3 9550 4380
> Fax: +61 3 9550 6125
> email: [log in to unmask]
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