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We've had pretty good success with TOTO3 from Molecular Probes which is easily excited by a 633 HeNe laser. One drawback is that it is a general nucleic acid stain so it tends to also bind RNA (with less affinity than to DNA)...but often we'll see some cytoplasmic staining as well as nuclear. This can be circumvented to a certain extent with RNAse treatment prior to applying the TOTO3. It also bleaches relatively fast...but can be preserved better if you mount with Molecular Probes Prolong Gold. Just keep in mind it's not DAPI--it won't be a bright or robust. But it does work.
We lived for many years with a confocal with no UV or 2p laser...so have quite a bit of experience trying to work out the kinks of TOTO3. Feel free to email me off list if you need more details.
Best,
d
Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945
415-209-2046
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]On
Behalf Of Zifu Wang
Sent: Tuesday, May 02, 2006 9:51 AM
To: [log in to unmask]
Subject: Re: DNA dye excited with visible laser
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello listers,
We have a Zeiss LSM 510 system without a UV laser or two-photon laser. I
would like to learn your suggestions on which DNA dye excited by visible
laser (Argon or HeNe) is the best substitute of DAPI.
Thanks.
-------------------------
Zifu Wang, Ph.D.
Optical Biology Core Facility
Developmental Biology Center
2114 McGaugh Hall
Univerity of California
Irvine, CA 92697-2275
Tel: (949) 824-3856
FAx:(949) 824-3571
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Guy Cox
Sent: Monday, May 01, 2006 10:45 PM
To: [log in to unmask]
Subject: Re: propidium iodide and death
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I'm not sure I can follow this argument. I certainly
cannot divide (nor indeed reproduce any other way since
I've had a vasectomy) and at 61 I'd say I'm pretty
much fully differentiated. But I've heard nobody
suggest that I'm not alive.
Guy
Joel Sheffield wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> You raise an interesting philosophical question. Is a mature nerve
> cell, or other fully differentiated cell, living or dead? I suppose
> that one could escape the paradox by stating that the "appropriate
> conditions" to support division of these cell types can not be found,
> but this seems more like an evasion than an answer. Perhaps the term
> itself is too vague, and we should look at the use of the term,
> "division competent" as a possibility. We should certainly accept
> the possibility that "living" might also mean "functional",
> "metabolically active", etc.
>
> Joel
>
>
>
> Date sent: Fri, 28 Apr 2006 14:29:25 -0400
> Send reply to: Confocal Microscopy List
<[log in to unmask]>
> From: "Robert J. Palmer Jr." <[log in to unmask]>
> Subject: propidium iodide and death
> To: [log in to unmask]
>
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>PI has a tradition in eukaryotic biology as an indicator of cell death
>>through membrane disruption. I am interested in whether any list
>>watchers can tell me of cases in which strong PI staining is not an
>>indicator of cell death. If many examples of PI staining of growing
>>(or growth-capable) cells exist, then of what is PI staining
>>indicative other than a (transient) membrane permeability difference
>>compared to untreated controls? FYI, my definition of cell death is
>>the inability of cells to divide when provided with appropriate
>>conditions. I am less interested in other measurements of viability -
>>"dividing or dead" is my motto... Thanks for your opinions..... Rob
>>Palmer -- Robert J. Palmer Jr., Ph.D. Natl Inst Dental Craniofacial
>>Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30,
>>Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax
>>301-402-0396
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [log in to unmask]
--
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
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