Re: combination of 3 fluorescence protein reporters

Posted by Catherine Degnin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/combination-of-3-fluorescence-protein-reporters-tp590861p590867.html

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Do you have suggestions on the best way to fix cells expressing  
mCherry-tagged proteins?  I get gorgeous images using live cells, but  
I lose a lot of signal using a fixation protocol that works  
beautifully with eGFP and YFP...

Thanks,
Catherine

On Nov 8, 2007, at 8:10 AM, Stephen Bunnell wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Xinyu,
>
>     We have had good luck with that combination, as well as
> mCerulean/mYFP/mCherry and mCerulean/mYFP/mStrawberry. Both of the  
> latter
> sets work well on our fixed dichroic Ultraview spinning disk  
> system, which
> has a custom built quad-pass dichroic that we acquired from Chroma.  
> We have
> no detectable bleedthough between these three channels using  
> 442/514/568 nm
> laser lines for excitation. By the book, Cherry looks like it  
> should be
> brighter and more stable, but Strawberry 'fits' our existing filter  
> sets
> (based on DsRed) much better.
>
>     I had expected that mVenus should be much better that YFP, but  
> in my
> hands (and filter sets) it did not offer any significant  
> advantages. It
> actually bleached _faster_ than mYFP under constant 514nm  
> illumination.
>
>     Best regards,
>
>     -Steve
>
> **********************************************************************
> ******
> Stephen C. Bunnell, Ph.D.
> Assistant Professor
> Tufts University Medical School
> Department of Pathology
> Jaharis Bldg., Room 512
> 150 Harrison Ave.
> Boston, MA 02111
>
> Phone: [phone]
> Fax:   [phone]
> Email: [hidden email]
>
>
> On 11/8/07 10:06 AM, "Xinyu Zhao" <[hidden email]> wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear listers,
>>
>> One of our users wanted to image CFP and YFP labeled cells and  
>> would like to
>> add a third fluorecence protein reporter later. I was wondering if  
>> anybody
>> could make a recomendation on this third one.
>>
>> Anybody has ever used a  CFP/YFP/mCherry combination ?
>>
>> Thank you very much.
>>
>>
>> Xinyu Zhao
>> Biomedical Imaging Core Lab
>> School of Medicine
>> University of Pennsylvania
>> Tel:  [phone]