sorry
Locked
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yesterday I demonstrated my distaste for a web site used in a thread by repeating something distasteful from it. I apologize to all who were offended by this approach. That thread had spun out of control as did my fingers on the keyboard. In the future I hope to refrain from adding to the political diatribe that inevitably makes its way onto this (and most other) scientific listservs. 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 |
 
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Steffen Dietzel |
DiD / DiI / DiO versions 'solid' and 'oil'
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello there, the DiI / DiO related membrane stain DiD from Molecular Probes comes in two flavors (as do its relatives): 'DiD' oil = DiIC18(5) oil (cat# D307) or as 'DiD' solid = DiIC18(5) solid (cat# D7757). Can anyone comment on what the advantages/disadvantages of the two flavors are? The idea for the experiment is to use DiD to stain the membranes of endothelial cells of blood vessels (from the interior of the vessel), to be able to distinguish the endothelial cells better from neighboring smooth muscle cells (SMCs) Povided that the endothelium is intact, the dye should not be able to reach the SMCs - or so ist the hope. DiD should be sufficiently in the red to allow the simultaneous usage of the green channel e.g. for Fluo-4 Ca measurements. Regards Steffen -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 skype: steffendietzel e-mail: [hidden email] |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Everyone When is a new version of a confocal operating system an "upgrade" and when it is a mere bug-fix?? Considering the high cost of buying any of the major manufacturer's systems, my view is that the software should work properly, with some allowance for unforeseen glitches that sneak through the pre-release testing. After all, the operation of the microscope is entirely dependent on the satisfactory implementation of the software as the interface between the user and the hardware. Consequently, I have considered (perhaps unreasonably...) that when you buy the system, you get bug-fixes to the software for free (especially, but not only, if you find, document and report the bugs yourself). On the other hand, I am quite willing to pay for a software upgrade that offers new capabilities that were not present in a previous version of the operating system. How then should we respond to vendors that offer new versions of operating systems that are no more than bug-fixes as "upgrades" and then expect us to pay out serious money to acquire them? I'd be interested in your responses, off list if necessary... thanks IAN * * * * * * * * * * * Prof Ian Gibbins Anatomy & Histology Flinders University GPO Box 2100 Adelaide SA 5001 AUSTRALIA [hidden email] voice: +61-8-8204 5271 fax: +61-8-8277 0085 http://som.flinders.edu.au/FUSA/Anatomy/ http://www.flinders.edu.au/neuroscience |
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Sylvie Le Guyader-2 |
Re: DiD / DiI / DiO versions 'solid' and 'oil'
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In reply to this post by Steffen Dietzel
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Steffen From my work in Zebrafish I'd recommend to purchase DiD solid so you can dissolve it in what you want. It might be difficult to get it to mix well in blood if it is diluted in oil. In live fish we were using DiI and DiO diluted about 5% in Ethanol. You'll definitely get some dye leaks to neighbouring cells after a few hours so you will need to image fairly quickly. For fixed fish we were using a saturated solution of DiI in chloroform. Leaks also occur but it takes several days at 4º. If you get the solid form, you can try a few things and see what works best. Powders are kept in a dry box. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 Huddinge Sweden +46 (0)8 608 9269 > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Steffen Dietzel > Sent: 15 July 2008 19:13 > To: [hidden email] > Subject: DiD / DiI / DiO versions 'solid' and 'oil' > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello there, > > the DiI / DiO related membrane stain DiD from > Molecular Probes comes in two flavors (as do its relatives): > > 'DiD' oil = DiIC18(5) oil (cat# D307) or as 'DiD' > solid = DiIC18(5) solid (cat# D7757). > > Can anyone comment on what the > advantages/disadvantages of the two flavors are? > > The idea for the experiment is to use DiD to > stain the membranes of endothelial cells of blood > vessels (from the interior of the vessel), to be > able to distinguish the endothelial cells better > from neighboring smooth muscle cells (SMCs) > Povided that the endothelium is intact, the dye > should not be able to reach the SMCs - or so ist > the hope. DiD should be sufficiently in the red > to allow the simultaneous usage of the green > channel e.g. for Fluo-4 Ca measurements. > > Regards > > Steffen > > -- > ----------------------- > Steffen Dietzel, PD Dr. rer. nat > Ludwig-Maximilians-Universität München > Walter-Brendel-Zentrum für experimentelle Medizin (WBex) > > Mail room (for letters etc.): > Marchioninistr. 15, D-81377 München > > Building location and address for courier, parcel services etc: > Marchioninistr. 27, D-81377 München (Großhadern) > > Phone: +49/89/2180-76509 > Fax-to-email: +49/89/2180-9976509 > skype: steffendietzel > e-mail: [hidden email] |
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In reply to this post by ian gibbins
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Ian, if your system doesn't perform as it should be and the issue is located on software level, then this should be fixed by an update for free - if the system is under warranty or covered by a service contract. Regarding upgrades, which add additional features, this you usually have to pay, unless something else was negotiated during the ordering (which might make sense). Michael > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello Everyone > > When is a new version of a confocal operating system an "upgrade" and > when it is a mere bug-fix?? > > Considering the high cost of buying any of the major manufacturer's > systems, my view is that the software should work properly, with some > allowance for unforeseen glitches that sneak through the pre-release > testing. After all, the operation of the microscope is entirely > dependent on the satisfactory implementation of the software as the > interface between the user and the hardware. Consequently, I have > considered (perhaps unreasonably...) that when you buy the system, you > get bug-fixes to the software for free (especially, but not only, if > you find, document and report the bugs yourself). On the other hand, I > am quite willing to pay for a software upgrade that offers new > capabilities that were not present in a previous version of the > operating system. > > How then should we respond to vendors that offer new versions of > operating systems that are no more than bug-fixes as "upgrades" and > then expect us to pay out serious money to acquire them? > > I'd be interested in your responses, off list if necessary... > > thanks > > IAN > > > > * * * * * * * * * * * > Prof Ian Gibbins > Anatomy & Histology > Flinders University > GPO Box 2100 > Adelaide SA 5001 > AUSTRALIA > > [hidden email] > voice: +61-8-8204 5271 > fax: +61-8-8277 0085 > > http://som.flinders.edu.au/FUSA/Anatomy/ > http://www.flinders.edu.au/neuroscience |
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In reply to this post by ian gibbins
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >How then should we respond to vendors that offer new versions of >operating systems that are no more than bug-fixes as "upgrades" and >then expect us to pay out serious money to acquire them? > >I'd be interested in your responses, off list if necessary... This is SOP. <sarcasm>If you don't like it, build your own microscope!</sarcasm> ____________________________________________________________________________ Michael Cammer, Senior Light Microscopist, Analytical Imaging Facility, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 URLs: microscopy http://www.aecom.yu.edu/aif/ and art http://coxcammer.com/ |
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Ignatius, Mike |
Re: DiD / DiI / DiO versions 'solid' and 'oil'
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In reply to this post by Sylvie Le Guyader-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I concur Sylvie response - I also would be concerned that any holes in the endothial layer would allow oil version to leak out. Better containment with the paste - applied with glass needles. To make protocol optimization a tad easier, we offer two sampler kits (below). For details our web site is best, rather than blogging here. Lipophilic Tracer Sampler Kit The Lipophilic Tracer Sampler kit contains 1 mg samples of nine different membrane stains. SKU# L-7781 This has more than just the DiI versions in case they work out better. But for just paste versions: (Ignore neuro - can work for any cell type.) NeuroTrace® Multicolor Tissue-Labeling Kit - DiO, DiI, DiD pastes, 500 mg each SKU# N-22884 Mike Ignatius Molecular Probes/Invitrogen -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader Sent: Wednesday, July 16, 2008 1:01 AM To: [hidden email] Subject: Re: DiD / DiI / DiO versions 'solid' and 'oil' Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Steffen From my work in Zebrafish I'd recommend to purchase DiD solid so you can dissolve it in what you want. It might be difficult to get it to mix well in blood if it is diluted in oil. In live fish we were using DiI and DiO diluted about 5% in Ethanol. You'll definitely get some dye leaks to neighbouring cells after a few hours so you will need to image fairly quickly. For fixed fish we were using a saturated solution of DiI in chloroform. Leaks also occur but it takes several days at 4º. If you get the solid form, you can try a few things and see what works best. Powders are kept in a dry box. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 Huddinge Sweden +46 (0)8 608 9269 > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Steffen Dietzel > Sent: 15 July 2008 19:13 > To: [hidden email] > Subject: DiD / DiI / DiO versions 'solid' and 'oil' > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello there, > > the DiI / DiO related membrane stain DiD from > Molecular Probes comes in two flavors (as do its relatives): > > 'DiD' oil = DiIC18(5) oil (cat# D307) or as 'DiD' > solid = DiIC18(5) solid (cat# D7757). > > Can anyone comment on what the > advantages/disadvantages of the two flavors are? > > The idea for the experiment is to use DiD to > stain the membranes of endothelial cells of blood > vessels (from the interior of the vessel), to be > able to distinguish the endothelial cells better > from neighboring smooth muscle cells (SMCs) > Povided that the endothelium is intact, the dye > should not be able to reach the SMCs - or so ist > the hope. DiD should be sufficiently in the red > to allow the simultaneous usage of the green > channel e.g. for Fluo-4 Ca measurements. > > Regards > > Steffen > > -- > ----------------------- > Steffen Dietzel, PD Dr. rer. nat > Ludwig-Maximilians-Universität München > Walter-Brendel-Zentrum für experimentelle Medizin (WBex) > > Mail room (for letters etc.): > Marchioninistr. 15, D-81377 München > > Building location and address for courier, parcel services etc: > Marchioninistr. 27, D-81377 München (Großhadern) > > Phone: +49/89/2180-76509 > Fax-to-email: +49/89/2180-9976509 > skype: steffendietzel > e-mail: [hidden email] |
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Steffen Dietzel |
Re: DiD / DiI / DiO versions 'solid' and 'oil'
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Mike, thanks for your response. Since this is going to be pretty specialized, I reply off list. First, I also asked a similar question via [hidden email], so the orignal question may end up in your inbox twice. Second, the tracer sampler kit would be nice if it weren't for the Ca-detectors I also have to use. So I have to stay clear of the green and preferably orange channels, that's why I am looking (only) into DiD in the first place. With the pastes: We want to put the dye into intact blood vessels, diameter maybe 100-200 µm. I don't think we are going to manage to stuff the paste inside. What do you think? We can let solutions flow through it via glass capilaries that are attached to the ends, so I planed to let some buffered solution flow in, incubate for let's say 30 min and then wash again with buffer before fluorescence microscopic observation. Does that sound like a doable experiment for you, regarding the DiD staining? Since I didn't find much details about the differences of the oil and solid versions, I brought it to the confocal list. As a side note, the DiD paste ( N22882) dosn't seem to have an entry on the web site and thus no price information. thanks for your help in this Steffen At 20:28 16.07.2008, you wrote: Search the CONFOCAL archive at |
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George McNamara |
Re: DiD / DiI / DiO versions 'solid' and 'oil'
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Steffen, Check out Ravnic et al 2005 Microvasc Res 70: 90, Pubmed 16095629: "The fluorescent dye 1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate was obtained from Sigma (St. Louis, MO). The carbocyanine dye was dissolved in ethanol (6 mg/ml) and stored as a 6.42 mM stock solution at 4- C. Immediately prior to infusion, the stock solution was diluted in phosphate buffered saline (PBS) containing glucose (200 mM) for a final concentration of 0.128 mM." Was used to label mouse vessels. The inventor of the technique, Rong Wen, has a paper submitted to Nature Protocols, so please keep an eye out for it (shows the entire endothelial cell takes up the dye and has a terrific image of a sprouting cell). Rong told me that diluting the stock immediately before is critical. Glucose is better than no glucose. Rong usually asphyxiates (spelling?) the mouse with CO2, but a smaller amount of DiI can be injected for live mouse imaging, in which case it mostly coats RBCs. Rong like the Sigma-Aldrich dye (less pure) because it is less expensive ($0.50/mouse) than Invitrogen's. I have not checked what other DiFamily members Sigma-Aldrich has. I also have not calculated how expensive the 1:1 Alexa Fluor 647-streptavidin : biotin-tomato lectin mix used in my upcoming Paddock 2.0 book chapter is, but Rong's DiI is a lot less expensive. Brant Weinstein used to inject zebrafish blood vessels with dyes, though he has mostly switched to fluorescent proteins, as in: Kamei M, Weinstein BM. Long-term time-lapse fluorescence imaging of developing zebrafish. Zebrafish. 2005;2(2):113-23. PMID: 18248171. See also his web site at NIH. Best wishes, George At 04:17 AM 7/17/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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