Re: Nuclear staining - Vendor Response

Posted by Casalini Patrizia on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Nuclear-staining-tp2191386p2202248.html

Dear Jason,

 

Thank you for your response, actually I did the control you mentioned and no contribution of autofluorescence has been detected; about dxr, it is a fluorescent compound which emits in red.

I performed nuclear staining with YOYO1 several times and had no problem, however the excitation in the red channel has been usually done with HeNe (543 nm); the difference in this experiment was only that to detect dxr, based on the United States patent 4,906,100, I need to excite with Ar 488 (Abs peak around 470-500 nm) and to collect in the red channel (em peak around 570nm).

First I though it was an interaction between dxr and DNA stains, however it seems not the case, since the signal is still present also in dxr untreated samples.

 

Probably there is something I miss, but I can’t imagine what.

 

Any suggestions are welcome!

 

Patrizia

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kilgore, Jason
Sent: giovedì 22 gennaio 2009 19.36
To: [hidden email]
Subject: Re: Nuclear staining - Vendor Response

 

 

** VENDOR RESPONSE **

 

Dear Patrizia,

 

Are you certain this is not due to autofluorescence contributed by the cells or the doxorubicin?  If you haven’t yet, I would suggest trying to image a no-dye control and see if it gives off a fluorescent signal at that wavelength.

 

Jason

 

Jason A. Kilgore

Molecular Probes Tech Support

 

 

 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Casalini Patrizia
Sent: Wednesday, January 21, 2009 1:46 AM
To: [hidden email]
Subject: Nuclear staining

 

Dear all,

 

I have to detect Doxorubicin in frozen tissue tumors from treated vs untreated mice. I counterstained nuclei either with YOYO-1 after permeabilization with 0.1% Triton or with SYBRGreen-1 with no permeabilization.

 

I set up sequential acquisition method with confocal BioRad2000 system as follows:

 

PMT1 Ex= 488; Em = BP 515/30.

PMT2 Ex= 488; Em = BP 600/50.

 

Could someone explain me why I still detect a strong signal with PMT2, even though there is the emission BP filter, in both cases (YOYO1 or SYBR green)? Given that the signal is present also in untreated samples, it can be due only to nuclear staining, can’t it?  

 

Thanks,

Patrizia

 

.......................................................................

Dr. Patrizia Casalini

Molecular Biology Unit

Experimental Oncology Dept.

Fondazione IRCCS Istituto Nazionale dei Tumori

Via Venezian, 1. 20133 Milano - Italy

Phone: [phone], Fax: [phone]

E-mail: [hidden email]

 

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