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Hi Renato,
Don't waste clean image analysis on bad samples!
It sounds like your sample preparation, immunostaining and/or imaging
conditions are highly variable. How about locking down these conditions.
and satisfying yourself that you have consistent imaging conditions. If
you have two very good monoclonal antibodies that bind far apart on the
same protein (specifically, farther than efficient FRET distance, or use
fluorophores with large spectral separation to minimize spectral
overlap, such as Alexa Fluor 488 and 647 ... hopefully without
introducing chromatic aberrations!), you should be able to find
conditions where the distribution patterns are consistent. If any of
your favorite proteins have a fluorescent protein tag, you can also take
advantage of it.
Effective permeabilization of nuclei is more difficult than
permeabilizing the plasma membrane. I believe nucleoli are especially
high density structures - may be difficult to get full size antibodies
to penetrate efficiently into them (assuming you have good nuclear
permeabilization).
Since you are fixing and permeabilizing the cells, you have your choice
of mounting media. I suggest you consider 2,2'-thiodiethanol for your
final mounting medium and use your best oil immersion objective lens.
See Staudt ... Hell 2007 (Staudt's PhD dissertation is available online
- might have more tips), along with Stan Vitha's listserv post on
gradual infiltration.
2,2'-thiodiethanol: a new water soluble mounting medium for high
resolution optical microscopy. </pubmed/17131355>Staudt T, Lang MC,
Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007
Jan;70(1):1-9.PMID: 17131355
There are plenty of literature on fluorescence colocalization. A few I
recommend include:
Multi-image colocalization and its statistical significance.
</pubmed/20858446> Fletcher PA, Scriven DR, Schulson MN, Moore ED.
Biophys J. 2010 Sep 22;99(6):1996-2005.PMID: 20858446
Quantifying colocalization by correlation: the Pearson correlation
coefficient is superior to the Mander's overlap coefficient.
</pubmed/20653013> Adler J, Parmryd I. Cytometry A. 2010
Aug;77(8):733-42.PMID: 20653013
Replicate-based noise corrected correlation for accurate measurements of
colocalization. </pubmed/18387047>Adler J, Pagakis SN, Parmryd I. J
Microsc. 2008 Apr;230(Pt 1):121-33.PMID: 18387047
Sincerely,
George
p.s. not specific for a single nucleolar protein, but I want to draw
your attention to this fascinating paper:
In vitro and intracellular production of peptide-encapsulated
fluorescent silver nanoclusters. </pubmed/17285671> Yu J, Patel SA,
Dickson RM. Angew Chem Int Ed Engl. 2007;46(12):2028-30. No abstract
available. PMID: 17285671
On 1/20/2011 6:03 AM, Renato Mortara wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Having being in the first of Jim Pawley's course I feel embarassed to ask
> this, but the doubt is haunting me. Here it goes:
>
> The system and approach: nucleoli of eukaryotic cells double immunolabeled
> for pairs of antigens. Z-sectioning through the structures (some quite
> spherical, others very disperse and granular) the fluorescence intensity
> profile of some of these pairs of antigens appear distinct, ie. the axial of
> distribution of different antigens varies among samples - that were NOT
> necessarily imaged or PREPARED on the same day.
>
> The question: is it valid to compare these distribution patterns
> (intensities along z axis) of antigen pairs between these different samples
> (nucleoli of cells A x cells B)?
>
> Many thanks !!
>
> Best
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: 55 11 5579-8306
> Fax: 55 11 5571-1095
> email: [log in to unmask]
> home page: www.ecb.epm.br/~ramortara
>
>
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