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Hi Patrick,

I agree that multiple tests need to be done in order to establish 
that protein localization is preserved during the various treatments.
Your point about the need for RNA as a structural element: since I am 
working with relatively small probes against enhancers, which seem 
not to have a lot of transcription units in them, I've been doing DNA 
FISH/protein stain combo with or without the RNAse treatment. I don't 
see dramatic changes in protein distribution with or without the 
RNAse treatment. However, this is on a very gross level, I agree that 
it is possible that some foci might change their location relatively 
to other nuclear structures or genes.
Same for the treatment with formamide: I don't see gross changes in 
protein distribution in my DNA/protein combo versus just protein 
immunodetection, but then again, the changes might be subtle.

One control I am trying to get to work right now is taking a 
well-identified transcription factor - regulatory sequence system, 
where the localization of the TF to its target sequence has been 
shown in vivo, and seeing that this localization can be detected via 
IF in my system. An example of such is Lac Repressor (LacI)-GFP 
fusion and its target sequence, multiple copies of Lac Operon sites 
(LacO). The visualization of LacI-GFP on LacO repeats has been 
described in a number of papers (for example, Vazquez et al (2006) 
Mol Bio Cell, where the clustering of LacI-GFP molecules on 64 LacO 
sites was very nicely shown).  I assume that, since Alexa fluors are 
brighter than GFP, I should be able to detect clustering of LacI-GFP 
on at least 64 LacO sites? I'd appreciate some feedback on this one 
from people who have experience with GFP detection in vivo and via IF 
.

Thank you,

Ella




>Hi,
>
>I think the combination of FISH and protein immuno localisation is a 
>ever lasting problem which needs multiple checks.
>A question in this is the presence of RNA as a linking and 
>structuring element in the cell nucleus. In order to reach high 
>specificity for FISH it is adviced to do a RNAase treatment, and 
>even some protease treatment.
>Further more, does a treatment with formamide not alter your epitope 
>and affects specificity of the immuno-detection?
>A fundamental question here is: how can we include the necessary 
>controls? Positive and negatives?
>I really follow this discussion with high interest.
>
>Patrick Van Oostveldt
>
>Quoting Ella Tour <[log in to unmask]>:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hi Carl,
>>
>>Thank you for mentioning the fixation/denaturation artifacts, I've been
>>wondering about that as well. I am familiar with the paper by Melan and
>>Sudler (also mentioned in Jason Swdlow's reply,
>>http://www.ncbi.nlm.nih.gov/pubmed/1527176). This paper demonstrates
>>that localization of injected FITC-labeled proteins changes as a result
>>of different methods of cell permeabilization, with worst artifacts
>>observed for protocols where permeabilization with TritonX was done
>>before fixation, the higher  TritonX concentrations, the worse the
>>redistribution. When cells were fixed first  (3.7% formaldehyde, 30
>>min) and then permeabilized (0.1% TritonX, 10 min), protein
>>distribution became less even and more punctate.
>>
>>Now, as far as I know, most immunofluorescent studies of nuclear
>>proteins employ the later protocol (2-4% formaldehyde fixation,
>>permeabilization with 0.1-0.3% TritonX). Many of these studies show
>>good colocalization between proteins belonging to the same nuclear
>>structures (localization of splicing factors to "speckles", for
>>example). So, at least for some proteins, the subnuclear localization
>>seems to be decently preserved under these conditions. Is there a
>>consensus as to whether this common fixation/permeabilization method
>>causes significant changes in protein localization within the nucleus?
>>
>>With regards to denaturation: often, we combine protein stains with DNA
>>in situ hybridization. The DNA FISH protocol calls for heating of the
>>tissue, in order to denaturate the embryonic DNA before the addition of
>>the DNA probe. So, before detecting the protein, I subject the embryos
>>to 15 min of 85 degrees Celsius and then an overnight hybridization in
>>50% formamide at 37 degrees Celsius. After that, we wash the probe with
>>solutions containing 0.3% CHAPS ( a zwitterionic detergent). Any red
>>flags here?
>>
>>Thank you,
>>
>>Ella
>>
>>>
>>>Hi 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

-- 




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
FAX 858-822-0460
email: [log in to unmask]