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re suggestion of fixation artefacts, can you provide a useful reference ?

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Thu 3/27/2008 01:11
To: [log in to unmask]
Subject: Re: Subnuclear localization of transcription factors in fixed tissue
 
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Re: Subnuclear localization of transcription factors in fiHi Ella,
One thing I didn't see mentioned in this thread was the possibility of aggregates or precipitates forming during fixation/denaturation.  It's the first thing I think of when I see the term "speckles" to describe a staining pattern.  A classic artifact studied for years was the large number of vessicles in endothelial cells seen in EM studies, the majority of which turned out to be derived from the fixation process.  
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
  ----- Original Message ----- 
  From: Ella Tour 
  To: [log in to unmask] 
  Sent: Wednesday, March 26, 2008 3:42 PM
  Subject: Re: Subnuclear localization of transcription factors in fixed tissue


  Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
  Dear Confocalists,

  Thanks a lot for your useful suggestions and the plenitude of great references. I am still digging through them.


  I will be definitely testing whether my protein foci colocalize with the markers for the known subnuclear structures, such as  the "speckles" (or "interchromatin granule clusters" in EM), Cajal  bodies and PML bodies.

  Some of you have raised a very valid point: that protein aggregates might be a result of an over-expression of a protein at abnormally high levels. This is not the case in our system: most of the times, we detect the endogenous protein in embryos. The antibodies we work with are specific, as they detect the Hox proteins in the regions where their genes are transcribed.

  Thank you very much again,

  Ella


-- 




  Ella Tour
  Department of Cell and Developmental Biology, 0349                     
  University of California, San Diego             
  9500 Gilman Drive, 4305 Bonner Hall                             
  La Jolla, CA  92093-0349
  Phone 858-822-0461
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  email: [log in to unmask]