help for oocyte staining method

John J. Lemasters lemaster at MED.UNC.EDU
Thu Nov 25 10:39:00 EST 1999


Dear Jian,

You want to stay away from DiOC6 to stain mitochondria. DiOC6 inhibits
mitochondrial respiration at concentrations of just a few nanomolar
(Mitochondrial dysfunction in lymphocytes from old mice: enhanced activation
of the permeability transition. Biochem Biophys Res Commun. 1997
;240:68-74.). You would be better off to use one of Leslie Loew's dyes, such
as tetramethyrhodamine methyester (TMRM). Load your cells in the range of
50-250 nM. I have a recent overview that might be helpful to you:

Lemasters, J.J., D.R. Trollinger, T. Qian, W.E. Cascio and H. Ohata (1999)
Confocal imaging of Ca2+, pH, electrical potential and membrane permeability
in single living cells.  In Methods in Enzymology, Vol. 302, Green
Fluorescent Protein, P.M. Conn, Ed., Academic Press, New York, pp. 341-358.

Good luck.

John J. Lemasters
Director, Cell and Molecular Imaging Facility
Department of Cell Biology & Anatomy
University of North Carolina at Chapel Hill
CB# 7090, 236 Taylor Hall
Chapel Hill, NC 27599-7090 USA
Tel: 919-966-5507
FAX: 919-966-7197
E-mail: lemaster at med.unc.edu


Jian Zhang wrote:

> Hi, every one,
>
> I prepare to do the mitochondrial staining of oocytes, but my result is
> not satisfied. At first I have problem on the medium making. The DiOC6 I
> used is not dissolved in the Eagle's medium well and when I filtered it,
> some pellets left on the filter. I want to know how to make 0.5 uM of
> staining medium for mitochondrial of oocytes and what solvents will be
> used. Also it will be great if the detail method can be provided,
> including how to stain.  Thank you very much.
>
> jian Zhang
> --
> Jian Zhang
> jianzhan at iastate.edu
> 2351 Kildee Hall
> Iowa State University
> Ames, Iowa, USA, 50011





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