Re: Keeping IMF slides good for long period?

Posted by Stephen Cody-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/CYTO2011-Pre-congress-courses-announcement-Fundamentals-of-Image-Cytometry-before-ISAC-2011-in-Baltie-tp6322453p6332841.html

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Dear Damir,

I can second Martin's suggestion. I've had Alexa488, Alexa546 and Cy3 (not
on the same slide) antibody stained cultured cells mounted in DPX for over
20 years. I just keep them in a box so the sun doesn't shine on them. You
may need to dehydrate gently to prevent cells flattening out, and infiltrate
with xylene prior to mounting. The cells were 4% PFA fixed.

If you need a reference (there may be others):
Cody, S.H., Williams, D.A. Optimizing confocal microscopy for thick
biological specimens. *In: Fluorescent and luminescent probes for biological
activity - A practical guide to technology for quantitative real-time
analysis* (ed W.T. Mason) Academic Press Limited, U.K. 2nd edition. Chapter
27 pp 350- 360. (1999).

Cheers
Steve


On 4 May 2011 05:07, Martin Wessendorf <[hidden email]> wrote:

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>
> Dear Damir--
>
>
> On 5/3/2011 12:58 PM, Damir Sudar wrote:
>
> I would really appreciate some good suggestions how to keep
>> immunofluorescently labeled slides of mammalian cells good for long
>> periods (3-5 months). The fluors are typically Alexa dyes or similar. Is
>> freezing at -20 or -80 a good idea? Any preparation tricks that keep the
>> fluorescence and the localization intact?
>>
>
> We stain tissue sections but I think our method should work for cells as
> well.  We stain with Cy2, Cy3, rhodamine and Cy5-labeled secondaries (and in
> the old days, Bodipy) and/or DNA dyes--but NOT fluorescein. After staining,
> we dehydrate the tissue in graded alcohols, clear in xylene and mount with
> DPX.  The DNA dyes tend to photobleach but photobleaching of the secondaries
> is acceptably low for our purposes.
>
> I have fluorescently stained sections that I processed that way almost 20
> years ago and have kept on my desk top in the intervening time, that I still
> use for teaching purposes.  I like having a solid mounting medium that
> doesn't require cold-storage.
>
> Good luck!
>
> Martin Wessendorf
>
>
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Stephen H. Cody