Secondary antibody for 405nm laser line ?
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lechristophe |
Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear list,
I would like to use all four lines of our new confocal for immunolabeling (405,488,532,633). For the moment I use Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth antibody labeling using the 405 channel instead of Hoechst. What secondary fluorescent antibody would you recommend ? Is there an Alexa405 or something similar that would be bright and stable ? Thanks a lot, Christophe Leterrier Postdoc INSERM UMR641 Neurobiology of ionic channels IFR Jean Roche - Mediterranee University Marseille, France |
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Claudia Campos |
Re: Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Christophe Leterrier escreveu: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France Molecular probes has Alexa 405, but I have never used it. Cheers Claudia Gonzalez lab University of Geneva Switzerland |
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Alison J. North |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal One of our users tried AlexaFluor 405 with the 405 line of our confocal and said it was absolutely gorgeous (I quote!). Best, Alison Christophe Leterrier wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France -- Alison J. North, Ph.D., Assistant Research Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 |
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Ramshesh, Venkat K |
Second Charleston Workshop on LIGHT MICROSCOPY FOR THE BIOSCIENCES (LMB)
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In reply to this post by Claudia Campos
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Second Charleston Workshop on LIGHT MICROSCOPY FOR THE BIOSCIENCES (LMB) Medical University of South Carolina May 18-23, 2008 The Second Charleston Workshop on LIGHT MICROSCOPY FOR THE BIOSCIENCES (LMB) Workshop provides a solid introduction to the concepts and practical applications of light microscopy relevant to modern cell and molecular biology. Students will have opportunities for extensive hands-on experience with state-of-the-art equipment for optical imaging, digital image processing, and fluorescence and confocal/multiphoton microscopy guided by experienced academic and commercial faculty. Lectures and laboratory exercises will include: optics of image formation; microscope alignment; phase contrast and differential interference contrast microscopy; video and digital cameras; contrast enhancement by analog and digital image processing; principles of fluorescence and fluorescence microscopy; ion imaging and fluorescent probes, including green fluorescent protein; fluorescence resonance energy transfer; and laser scanning confocal and multiphoton microscopy. A commercial faculty representing leading microscope manufacturers will make available for student use the latest and most advanced instrumentation for light microscopy, image detection and computerized image analysis. The LMB Workshop is designed for doctoral level scientists, advanced pre-doctoral students and high level technical personnel. No prior experience with microscopy is required. All students will benefit from in-depth interaction with instructors. Students are encouraged to bring their own specimens for analysis. Tuition: $750.00 Application Deadline: April 1, 2008 Principal Instructors: John J. Lemasters, M.D., Ph.D., Organizer P. Darwin Bell, Ph.D. Margaret Kelly, Ph.D. Peter Komlosi, Ph.D. Anna-Liisa Nieminen, Ph.D. Venkat Ramshesh, Ph.D. To apply, send a curriculum vita and a brief letter describing your research interests and reasons for enrolling. Because the course is expected to be oversubscribed, applicants should inquire as soon as possible. Please indicate your complete mailing address, telephone/fax number and email address. Full consideration will be given to applications received by April 1, 2008. For further information or to apply, contact: Venkat K Ramshesh Medical University of South Carolina Center for Cell Death, Injury and Regeneration and Hollings Cancer Center 280 Calhoun Street, PO Box 250140 Charleston, SC 29425 Telephone (843) 792- 3530, FAX (843) 792-1617 E-mail: [hidden email] |
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Bjornsson, Chris |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Christophe, I've had great luck with Alexa405, and highly recommend using this instead of Hoechst, which has a broad enough emission it can be problematic even for spectral imaging and linear unmixing. If you still want a channel for nuclei, CyQuant works great. I'm imaging a sample with Alexa405 right now actually; the only caution is that it seems to photobleach more quickly than my other 4 labels; but I'm trying to burn through 80-micron thick tissue slices, so this may not be typical... Cheers, Chris Chris Bjornsson Director, Microscopy & Imaging Research Core Center for Biotechnology and Interdisciplinary Studies, Room 2149 Rensselaer Polytechnic Institute 110 8th St., Troy, NY 12180 ph. 518-276-3456 -----Original Message----- From: Confocal Microscopy List on behalf of Christophe Leterrier Sent: Wed 3/5/2008 10:45 AM To: [hidden email] Subject: Secondary antibody for 405nm laser line ? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, I would like to use all four lines of our new confocal for immunolabeling (405,488,532,633). For the moment I use Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth antibody labeling using the 405 channel instead of Hoechst. What secondary fluorescent antibody would you recommend ? Is there an Alexa405 or something similar that would be bright and stable ? Thanks a lot, Christophe Leterrier Postdoc INSERM UMR641 Neurobiology of ionic channels IFR Jean Roche - Mediterranee University Marseille, France |
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simon walker (BI) |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
We have used Alexa Fluor 405 (Invitrogen)
with limited success. Our experience of it is that it’s reasonably
bright, but bleaches very quickly both under epi-illumination and with confocal
lsm. Mounting with an antifade seems to help (we’ve used
Vectasheild), but it still bleaches very quickly relative to, for example,
AF488. If someone has found a bright and stable 405-excitable fluorescent
dye I would be very interested to know. Perhaps Qdots maybe a possible
alternative? Simon Babraham Institute UK From:
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
What about Lucifer yellow? It is excitable at 405 but emission is in yellow. With AOBS and sequential scanning mode it should be theoretically distinguishable from others green, yellow and red dyes. But I have never work with that in this way, so I don’t know exactly.
Sincereley Ondrej -------------------------------------------- Ondřej Šebesta Faculty of Science, Charles University in Prague Vinicna 7 128 44 Prague Czech Republic
Phone: +420 2 2195 1943 e-mail: [hidden email]
-----Original Message-----
We have used Alexa Fluor 405 (Invitrogen) with limited success. Our experience of it is that it’s reasonably bright, but bleaches very quickly both under epi-illumination and with confocal lsm. Mounting with an antifade seems to help (we’ve used Vectasheild), but it still bleaches very quickly relative to, for example, AF488. If someone has found a bright and stable 405-excitable fluorescent dye I would be very interested to know. Perhaps Qdots maybe a possible alternative? Simon Babraham Institute Cambridge UK
From:
Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, |
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Asson-Batres, Mary Ann |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by simon walker (BI)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Out of curiosity, what is the problem you are having with Hoechst? Or are you looking for a 405 marker label to improve specificity? Mary Ann Tennessee State University Nashville, TN USA ________________________________ From: Confocal Microscopy List on behalf of simon walker (BI) Sent: Wed 3/5/2008 10:17 AM To: [hidden email] Subject: Re: Secondary antibody for 405nm laser line ? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have used Alexa Fluor 405 (Invitrogen) with limited success. Our experience of it is that it's reasonably bright, but bleaches very quickly both under epi-illumination and with confocal lsm. Mounting with an antifade seems to help (we've used Vectasheild), but it still bleaches very quickly relative to, for example, AF488. If someone has found a bright and stable 405-excitable fluorescent dye I would be very interested to know. Perhaps Qdots maybe a possible alternative? Simon Babraham Institute Cambridge UK ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier Sent: 05 March 2008 15:46 To: [hidden email] Subject: Secondary antibody for 405nm laser line ? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, I would like to use all four lines of our new confocal for immunolabeling (405,488,532,633). For the moment I use Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth antibody labeling using the 405 channel instead of Hoechst. What secondary fluorescent antibody would you recommend ? Is there an Alexa405 or something similar that would be bright and stable ? Thanks a lot, Christophe Leterrier Postdoc INSERM UMR641 Neurobiology of ionic channels IFR Jean Roche - Mediterranee University Marseille, France |
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lechristophe |
Re: Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I have non problem with Hoechst, I use it for DNA/Nucleus labeling and it works very well. It's just that I would like to use the 405 channel to label something else (not the DNA) with an antibody, so I'm looking for a fluorescent secondary antibody suitable for 405nm exitation.
Christophe On Wed, Mar 5, 2008 at 5:35 PM, Asson-Batres, Mary <[hidden email]> wrote: Out of curiosity, what is the problem you are having with Hoechst? Or are you looking for a 405 marker label to improve specificity? |
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Michael Weber-4 |
Re: Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Christophe, unfortunately I have to agree that Alexa 405 bleaches rather fast. So far I am not aware of other commercial secondary antibody for 405 nm. Michael Christophe Leterrier wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I have non > problem with Hoechst, I use it for DNA/Nucleus labeling and it works > very well. It's just that I would like to use the 405 channel to label > something else (not the DNA) with an antibody, so I'm looking for a > fluorescent secondary antibody suitable for 405nm exitation. > > Christophe > > On Wed, Mar 5, 2008 at 5:35 PM, Asson-Batres, Mary > <[hidden email] <mailto:[hidden email]>> wrote: > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Out of curiosity, what is the problem you are having with Hoechst? > Or are you looking for a 405 marker label to improve specificity? > > Mary Ann > Tennessee State University > Nashville, TN USA > > ________________________________ > > From: Confocal Microscopy List on behalf of simon walker (BI) > Sent: Wed 3/5/2008 10:17 AM > To: [hidden email] <mailto:[hidden email]> > Subject: Re: Secondary antibody for 405nm laser line ? > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We have used Alexa Fluor 405 (Invitrogen) with limited success. Our > experience of it is that it's reasonably bright, but bleaches very > quickly both under epi-illumination and with confocal lsm. Mounting > with an antifade seems to help (we've used Vectasheild), but it > still bleaches very quickly relative to, for example, AF488. If > someone has found a bright and stable 405-excitable fluorescent dye > I would be very interested to know. Perhaps Qdots maybe a possible > alternative? > > Simon > > Babraham Institute > > Cambridge > > UK > > > > ________________________________ > > From: Confocal Microscopy List [mailto:[hidden email] > <mailto:[hidden email]>] On Behalf Of Christophe > Leterrier > Sent: 05 March 2008 15:46 > To: [hidden email] <mailto:[hidden email]> > Subject: Secondary antibody for 405nm laser line ? > > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France |
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M. van de corput |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by simon walker (BI)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal For those not knowing, alexa fluor dyes are reported to bleach more quickly when embedded in vectashield. Invitrogen has ProLong (gold) antifading embedding medium that is more suitable for all alexa dyes. The bleaching is much less and the dyes are more stable over time. I have re-used slides that were almost 1 year old that were stored at 4oC. The dyes were less bright and bleached more quickly but for a year old slide I was surprised it was still that good. Pro-long gold needs curing overnight and at least 4 days of absolute curing. Only then the refractive index gets to 1.45. It is not wise to immediatly go look at the slides when just embedded. The signals will be weak and blurry. As for blue dyes. My experience with Alexa Fluor 350 is not that good. It works but for small targets the dyes are too weak as compared to Alexa488 or 594. For bigger/abundant targets it's fine. Mariette Mariette ____________________________________________ Dr. M.P.C. Kemner-van de Corput ____________________________________________ MGC - Dept. of Cell Biology & Genetics Erasmus Medical Center Dr. Molewaterplein 50, 3015 GE Rotterdam POB 2040, 3000 CA Rotterdam, The Netherlands Delivery adres: Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands Office: H-Ee751; tel: +31 10 704.3949 Lab: H-Ee710; tel lab: +31 10 704.3315 tel secr: +31 10 704.3169 ____________________________________________ http://www2.eur.nl/fgg/ch1/cellbiology/ http://www.thesis.kemner.biz/ ____________________________________________ Op Wo, 5 maart, 2008 5:17 pm, schreef simon walker (BI): > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We have used Alexa Fluor 405 (Invitrogen) with limited success. Our > experience of it is that it's reasonably bright, but bleaches very > quickly both under epi-illumination and with confocal lsm. Mounting > with an antifade seems to help (we've used Vectasheild), but it still > bleaches very quickly relative to, for example, AF488. If someone has > found a bright and stable 405-excitable fluorescent dye I would be very > interested to know. Perhaps Qdots maybe a possible alternative? > > Simon > > Babraham Institute > > Cambridge > > UK > > > > ________________________________ > > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Christophe Leterrier > Sent: 05 March 2008 15:46 > To: [hidden email] > Subject: Secondary antibody for 405nm laser line ? > > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France > > |
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simon walker (BI) |
Re: Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal It is not correct to say that ProLong Gold outperforms Vectasheild with alexa dyes. Invitrogen's own data show that Vectasheild outperforms ProLong Gold with Alexa dyes excitable at wavelengths of 488 nm and less. It is only the Alexa dyes that are excitable in the green and particularly the red part of the spectrum that significantly benefit from ProLong. Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mariette P.C. Kemner - van de Corput Sent: 06 March 2008 12:21 To: [hidden email] Subject: Re: Secondary antibody for 405nm laser line ? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal For those not knowing, alexa fluor dyes are reported to bleach more quickly when embedded in vectashield. Invitrogen has ProLong (gold) antifading embedding medium that is more suitable for all alexa dyes. The bleaching is much less and the dyes are more stable over time. I have re-used slides that were almost 1 year old that were stored at 4oC. The dyes were less bright and bleached more quickly but for a year old slide I was surprised it was still that good. Pro-long gold needs curing overnight and at least 4 days of absolute curing. Only then the refractive index gets to 1.45. It is not wise to immediatly go look at the slides when just embedded. The signals will be weak and blurry. As for blue dyes. My experience with Alexa Fluor 350 is not that good. It works but for small targets the dyes are too weak as compared to Alexa488 or 594. For bigger/abundant targets it's fine. Mariette Mariette ____________________________________________ Dr. M.P.C. Kemner-van de Corput ____________________________________________ MGC - Dept. of Cell Biology & Genetics Erasmus Medical Center Dr. Molewaterplein 50, 3015 GE Rotterdam POB 2040, 3000 CA Rotterdam, The Netherlands Delivery adres: Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands Office: H-Ee751; tel: +31 10 704.3949 Lab: H-Ee710; tel lab: +31 10 704.3315 tel secr: +31 10 704.3169 ____________________________________________ http://www2.eur.nl/fgg/ch1/cellbiology/ http://www.thesis.kemner.biz/ ____________________________________________ Op Wo, 5 maart, 2008 5:17 pm, schreef simon walker (BI): > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We have used Alexa Fluor 405 (Invitrogen) with limited success. Our > experience of it is that it's reasonably bright, but bleaches very > quickly both under epi-illumination and with confocal lsm. Mounting > with an antifade seems to help (we've used Vectasheild), but it still > bleaches very quickly relative to, for example, AF488. If someone has > found a bright and stable 405-excitable fluorescent dye I would be very > interested to know. Perhaps Qdots maybe a possible alternative? > > Simon > > Babraham Institute > > Cambridge > > UK > > > > ________________________________ > > From: Confocal Microscopy List [mailto:[hidden email]] > Behalf Of Christophe Leterrier > Sent: 05 March 2008 15:46 > To: [hidden email] > Subject: Secondary antibody for 405nm laser line ? > > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France > > |
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Benoit Labarthe |
Re: Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
hello,
we used AnnexinV conjugated to Pacific Blue, on a spinning disk confocal with reasonnable bleaching. benoit On Thu, Mar 6, 2008 at 7:30 AM, simon walker (BI) <[hidden email]> wrote: It is not correct to say that ProLong Gold outperforms Vectasheild with -- Benoît Labarthe, PharmD Institut de Cardiologie de Montréal Montreal Heart Institute Centre de Recherche, Laboratoire du Dr.Théroux 5000 Bélanger Est Montréal Québec Canada H1T 1C8 Tel (514)-376 3330 ext 3017 Fax (514)- 376 1076 |
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Bjornsson, Chris |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by M. van de corput
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I use ProLong and a handful of different Alexa dyes every time; the A405 still bleaches faster than the others, unfortunately! Cheers, Chris Chris Bjornsson Director, Microscopy & Imaging Research Core Center for Biotechnology and Interdisciplinary Studies, Room 2149 Rensselaer Polytechnic Institute 110 8th St., Troy, NY 12180 ph. 518-276-3456 -----Original Message----- From: Confocal Microscopy List on behalf of Mariette P.C. Kemner - van de Corput Sent: Thu 3/6/2008 7:21 AM To: [hidden email] Subject: Re: Secondary antibody for 405nm laser line ? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal For those not knowing, alexa fluor dyes are reported to bleach more quickly when embedded in vectashield. Invitrogen has ProLong (gold) antifading embedding medium that is more suitable for all alexa dyes. The bleaching is much less and the dyes are more stable over time. I have re-used slides that were almost 1 year old that were stored at 4oC. The dyes were less bright and bleached more quickly but for a year old slide I was surprised it was still that good. Pro-long gold needs curing overnight and at least 4 days of absolute curing. Only then the refractive index gets to 1.45. It is not wise to immediatly go look at the slides when just embedded. The signals will be weak and blurry. As for blue dyes. My experience with Alexa Fluor 350 is not that good. It works but for small targets the dyes are too weak as compared to Alexa488 or 594. For bigger/abundant targets it's fine. Mariette Mariette ____________________________________________ Dr. M.P.C. Kemner-van de Corput ____________________________________________ MGC - Dept. of Cell Biology & Genetics Erasmus Medical Center Dr. Molewaterplein 50, 3015 GE Rotterdam POB 2040, 3000 CA Rotterdam, The Netherlands Delivery adres: Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands Office: H-Ee751; tel: +31 10 704.3949 Lab: H-Ee710; tel lab: +31 10 704.3315 tel secr: +31 10 704.3169 ____________________________________________ http://www2.eur.nl/fgg/ch1/cellbiology/ http://www.thesis.kemner.biz/ ____________________________________________ Op Wo, 5 maart, 2008 5:17 pm, schreef simon walker (BI): > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > We have used Alexa Fluor 405 (Invitrogen) with limited success. Our > experience of it is that it's reasonably bright, but bleaches very > quickly both under epi-illumination and with confocal lsm. Mounting > with an antifade seems to help (we've used Vectasheild), but it still > bleaches very quickly relative to, for example, AF488. If someone has > found a bright and stable 405-excitable fluorescent dye I would be very > interested to know. Perhaps Qdots maybe a possible alternative? > > Simon > > Babraham Institute > > Cambridge > > UK > > > > ________________________________ > > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Christophe Leterrier > Sent: 05 March 2008 15:46 > To: [hidden email] > Subject: Secondary antibody for 405nm laser line ? > > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France > > |
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In reply to this post by Benoit Labarthe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have aTi:Sapphire laser system we are interested in selling. This is the mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.). It comes with a spectrometer. One of the diode lasers used in the Millenia is not operational: if you're interested, then we'd have that repaired. If this is of interest to you, please make an offer with the idea that the Millennia is repaired and in prime operating condition. Send email to: Jerry Sedgewick: [hidden email]. Thanks! Jerry Sedgewick Program Director, Biomedical Image Processing Lab University of Minnesota ÐÏࡱá --- http://USFamily.Net/dialup.html - $8.25/mo! -- http://www.usfamily.net/dsl.html - $19.99/mo! --- |
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Lenaldo Rocha |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Christophe I've read that cascade blue excited with 405 laser is as bright as Alexa488, that might be a good option. I don't have personal experience with it so can't tell if it fades faster than any other dye. Lenaldo Christophe Leterrier wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling (405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth > antibody labeling using the 405 channel instead of Hoechst. What > secondary fluorescent antibody would you recommend ? Is there an > Alexa405 or something similar that would be bright and stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France |
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Junya HIROI |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Christophe, I was also looking for a fluorescent dye for 405 nm excitation, and I compared Alexa 405 with Pacific Blue. Both dyes bleached to some extent with ProlongGold, but Pacific Blue was absolutely brighter than Alexa 405, when compared at the same dilution. So, I recommend Pacific Blue, although Molecular Probes sells Pacific Blue for flow cytometry. I found another problem on Alexa 405: it nonspecifically binds to nuclei ! I asked Molecular Probes/Invitrogen about this problem, and they recommended me to use their blocking solution, Image-iT FX Signal Enhancer. It actually worked well, but Pacific Blue does not have such a problem, so that is another reason I like Pacific Blue. Junya -- _____________________________________ Aäç ¹}AiãÈåw ðUw³º §216-8511 ìès{Oæ¶2-16-1 tel: 044-977-8111 àü3629 fax: 044-976-7083 e-mail: [hidden email] _____________________________________ -- |
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Fredrik Wermeling |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I´ve also used Pacific blue with good result. The target for the conjugated antibody I used has a very high expression which I guess makes it more easy to get a good signal. /fredrik
----- Original Message ----- > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear list, > > I would like to use all four lines of our new confocal for > immunolabeling(405,488,532,633). For the moment I use > Hoechst/Alexa488/Alexa555/Cy5, but I > would like to have a fourth antibody labeling using the 405 > channel instead > of Hoechst. What secondary fluorescent antibody would you > recommend ? Is > there an Alexa405 or something similar that would be bright and > stable ? > > Thanks a lot, > > Christophe Leterrier > > Postdoc > INSERM UMR641 Neurobiology of ionic channels > IFR Jean Roche - Mediterranee University > Marseille, France > |
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lechristophe |
Re: Secondary antibody for 405nm laser line ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Thank you all for your responses and recommandation. I ordered goat
anti-rabbit Alexa405 antibody and will keep you informed about its
performances. I'd be very interested in an anti-chicken secondary
antibody that can be excited at 405 nm (because I'm using a chicken
antibody to stain an abundant structural marker in my cells, keeping
the rabbit and mouse antibodies for more exotic targets). Unfortunately
Invitrogen/Molecular Probes does not sell anti-chicken secondaries
conjuguated to Alexa 405 or Cascade Blue :(
Christophe Leterrier |
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Glen MacDonald-2 |
Re: Secondary antibody for 405nm laser line ?
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In reply to this post by Junya HIROI
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal All of the Alexa fluor conjugates have a tendency to stick non- specifically via charge interactions of the anionic dye moiety. the Image-iT fx does helps greatly, especially in thick samples. In regard to using the blue fluorophores for the more prominent targets in the tissue, the human visual system is only weakly responsive to blue wavelengths and lacks blue-sensitive cones in the fovea, the region of greatest visual acuity. In general, if the smallest or rarest bits get the green, red or gray channel, they are noticeable with less histogram manipulation than if the smallest bits are viewed in the blue channel. Regards, Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On Mar 6, 2008, at 7:09 AM, Junya HIROI wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Christophe, > > I was also looking for a fluorescent dye for 405 nm excitation, and > I compared Alexa 405 > with Pacific Blue. Both dyes bleached to some extent with > ProlongGold, but Pacific Blue was > absolutely brighter than Alexa 405, when compared at the same > dilution. So, I recommend > Pacific Blue, although Molecular Probes sells Pacific Blue for flow > cytometry. > > I found another problem on Alexa 405: it nonspecifically binds to > nuclei ! I asked Molecular > Probes/Invitrogen about this problem, and they recommended me to > use their blocking > solution, Image-iT FX Signal Enhancer. It actually worked well, > but Pacific Blue does not have > such a problem, so that is another reason I like Pacific Blue. > > Junya > > -- > _____________________________________ > Aäç > ¹}AiãÈåw ðUw³º > §216-8511 ìès{Oæ¶2-16-1 > tel: 044-977-8111 àü3629 > fax: 044-976-7083 > e-mail: [hidden email] > _____________________________________ > > -- |
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