Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
We stain the plasmalemma of cells for confocal LM almost every day, and have gotten the best results using DiI, the classical dye for staining neurons & tracing pathways in the brain.
It excites with either blue or green light and emits yellow/red, and it NEVER bleaches. It also works on live or fixed cells, equally well.
(Only problem might be, if it stains the collagen gel. That I don't know.)
Write me directly for details, if you wish.
Good luck,
John H.
At 10:49 AM +0000 on 2/10/03, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear all,
>
>We are trying to image human fibroblasts live in
>collagen gels. While we have no problem with other
>types of cells, these fibroblasts are so huge that our
>standard marker (GFP) bleaches so fast that by the
>time we have gone through the whole cell volume, there
>is almost no fluorescence left (let alone for doing a
>time-lapse...). We have used both confocal and
>standard fluo/CCD imaging but even after all tricks
>tried (reducing the number of slices, of time points,
>minimal light exposure...), we are still limited to 2
>or 3 times the cell volume before the fluo dies. We
>have tried using a standard cell dye CFDA-SE, but with
>no better results. Would any one have any ideas on how
>to label these big cells (whole volume or at least
>membrane) with a more "unbleachable" approach?
>
>Cheers
>
>Maryse
>
>=====
>Maryse Bailly, Ph.D
>Division of Cell Biology, Institute of Ophthalmology
>University College London
>11-43 Bath Street, London, EC1V 9EL
>Tel: (44) 20 7608 4051
>Fax: (44) 20 7608 4034
>Email: [log in to unmask]
>
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--
Prof. John Heuser, M.D.
Dept. of Cell Biology Box 8228
Washington Univ. Sch. of Med.
660 South Euclid Ave.
St Louis, MO. 63110
work tele: 314-362-5105
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