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mayandi,
I guess counterstaining the membrane as well as the cytoplasm is
going to be the way to go.
Thanks,
Carol


>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Carol, based on the information you gave you can use either FM-123 or
>Propidum iodide for counter staining membranes. You can try the standard
>FDA (Fluoroscine diacetate) to both assess the live cells as well as the
>cytoplasm. These dyes works best for live cells. If you use fixed cells
>then we need to think something else.
>Shiv
>
>Mayandi Sivaguru, Ph.D.,
>Associate Director
>Molecular Cytology Core Facility
>Molecular Biology Program
>2, Tucker Hall
>University of Missouri
>Columbia, MO 65211-7400
>
>Voice: 1-573-882-4895
>Fax: 1-573-882-0123
>
>www.biotech.missouri.edu/mcc/
>
>
>At 04:11 PM 12/13/02 -0500, you wrote:
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hello,
>>I am helping someone determine whether their protein is on the
>>membrane, inside the cell, or both.  the living cells (I don't
>>remember what kind they are) are grown on cover slips and are very
>>flat.  It is difficult to tell.  they are thinking of counterstaining
>>with a cytoplasmic dye.  Several come to mind, like rhodamine 123 for
>>mitochondria, or a Cell Tracker dye.  conversely one could try to
>>stain the cell membrane.
>>Does anyone have any suggestions?  Would it be OK to use cells that
>>are rounding up? They are the brightest but I'm worried their
>>membranes may be compromised.
>>Thanks in advance.
>>Carol
>>--
>><><><><><><><><><><><><><><><><><><><><><><>
>>Carol Bayles
>>Microscopy,  Imaging & Fluorimetry (MIF)
>>Bioresource Center
>>160 Biotech Bldg
>>www.brc.cornell.edu
>>
>>Confocal and Multiphoton Microscopy
>>Nanobiotechnology Center
>>D21-H Clark Hall
>>www.nbtc.cornell.edu
>>
>>Cornell University
>>Ithaca NY 14853
>>607-254-4860
>>607-254-4847  fax


--
<><><><><><><><><><><><><><><><><><><><><><>
Carol Bayles
Microscopy,  Imaging & Fluorimetry (MIF)
Bioresource Center
160 Biotech Bldg
www.brc.cornell.edu

Confocal and Multiphoton Microscopy
Nanobiotechnology Center
D21-H Clark Hall
www.nbtc.cornell.edu

Cornell University
Ithaca NY 14853
607-254-4860
607-254-4847  fax