Re: Keeping IMF slides good for long period?

Posted by Boswell, Carl A - (cboswell) on
URL: http://confocal-microscopy-list.275.s1.nabble.com/CYTO2011-Pre-congress-courses-announcement-Fundamentals-of-Image-Cytometry-before-ISAC-2011-in-Baltie-tp6322453p6332819.html

Freezing (-20C) seems to work fine with all the preps I've seen in which this has been done.  It probably helps that the antifade mounting medium has glycerol in it.  Sealing the coverslip with nail polish or other hardening medium helps to limit dehydration, as well as secure it when cleaning or using under oil.  I've seen very nice neuromuscular junction staining (standard immunostaining protocols) that were several months old.
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, May 03, 2011 12:07 PM
To: [hidden email]
Subject: Re: Keeping IMF slides good for long period?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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


--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]