Re: Nuclear staining - Vendor Response

Posted by RICHARD BURRY on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Nuclear-staining-tp2191386p2203309.html

Patrizia
 
According to Hutun in Journal of Fluorescence 2004 14(2);217-222, the excitation spectra of doxorubicin is broad, including not only 488nm but also 543 and even  a little at 405 nm.  Emission is highest at 590 nm.  We are using the green HeNi (543 nm) and the 590 nm emission filter.  I do not know if this will help with the background, but try it!
 
Dick

----- Original Message -----
From: Casalini Patrizia <[hidden email]>
Date: Friday, January 23, 2009 3:56 am
Subject: Re: Nuclear staining - Vendor Response
To: [hidden email]

> 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 > 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       > 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: <A title=mailto:[e-mail] href="javascript:main.compose('new','t=[e-mail]')">patrizia.casalini@...   > AVVISO IMPORTANTE   > La presente comunicazione, che potrebbe contenere informazioni riservate e/o protette da segreto professionale, è indirizzata esclusivamente ai destinatari della medesima qui indicati. Ogni informazione qui contenuta, che non sia relativa alla nostra attività caratteristica, deve essere considerata come non inviata. Nel caso in cui abbiate ricevuto per errore la presente comunicazione, vogliate cortesemente darcene immediata notizia, rispondendo a questo stesso indirizzo di e-mail, e poi procedere alla cancellazione di questo messaggio dal Vostro sistema. E' strettamente proibito e potrebbe essere fonte di violazione di legge qualsiasi uso, comunicazione, copia o diffusione dei contenuti di questa comunicazione da parte di chi la abbia ricevuta per errore o in violazione degli scopi della presente. Ricordiamo che la tecnologia di trasmissione utilizzata non consente di garantire l’autenticità del mittente né l’integrità dei dati   > IMPORTANT NOTICE > This communication, that may contain confidential and/or legally privileged information, is intended solely for the use of the intended addressees. Every information or advice contained in this communication is subject to the terms and conditions provided by the agreement governing the engagement with such a client. If you have received this communication in error, please notify us immediately by responding to this email and then delete it from your system. Any use, disclosure, copying or distribution of the contents of this communication by a not-intended recipient or in violation of the purposes of this communication is strictly prohibited and may be unlawful. The transmission technology used to send this mail can’t grant neither the sender identity nor the data integrity     > P Rispetta l'ambiente: se non ti è necessario, non stampare questa mail. > P Before printing, think about the environment       > Spam > Not spam > Forget previous vote

Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
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