5 dye imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear all, I know this question comes up once per page, but could you share your favourite dye combination for the 5 colour fluorescent imaging? Lets say we're lazy and do not want to conjugate our AB; so which commercial dyes do you normally pick? I have today picked Alexa 488,555,594 and DyLight 680+DAPI. My Alexa 555 labeling will be extremely strong, so I am hoping to use a very narrow slit on an SP5. Alexa 488 and 555 will be cytoplasmic; 594 and 680 will be membrane. My considerations were: not to have membrane proteins stained in the autofluorescent range; to have the weakest staining in the cleanest channel (680). So do you advertise to users something more exotic on regular basis? Do you prophesy quantum dots or BV dyes? do you flip DAPI and put your proteins in the UV range? those will be frozen tissue sections. Thanks in advance! Marcia |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Marcia, Lots of Brilliant Violet's (BV###) available and you have spectral confocal microscope(s). https://www.bdbiosciences.com/documents/Horizon-Brilliant-Dyes-Booklet.pdf (couple of years old, BV and BUV only). I suggest 5plex BV tandems. All will also fluoresce to some extent at the BV421 emission peak. Ask your BD biosciences rep about BB515 vs other BB's for imaging. Plan "J": two state of the art fluorescent dyes commercially available are: Janelia Fluor 549 https://www.tocris.com/dispprod.php?ItemId=524906#.WMWGRjsrKUl Janelia Fluor 646 https://www.tocris.com/dispprod.php?ItemId=524907#.WMWGFDsrKUl see the web pages for "custom conjugation service by that vendor. Luke Lavis at Janelia Research Campus has even more fluorophores (and at HHMI, can give them out for free to academics who convence him you'll use them well), https://www.janelia.org/open-science/janelia-fluor-dyes the "square ringed" coumarin (0.96 QY, all the JF's have improved photostability too) in Grimm ... Lavis 2015 Nature Methods, for example. An alternative family of polymers to the Brilliant's is ThermoFisher's Super Bright Polymer Dyes (I would have called them Super Duper Bright Dyes, ThermoFisher should hire me to be their cytometry products marketing namer), https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/super-bright-polymer-dyes.html Super Bright 436 Super Bright 600 enjoy, George p.s. a lot of dyes photobleach quickly. One way to tell a biochemist/molecular biologist from a fluorescence microscopist, is the former go back to buckets/Illumina sequencers, the latter get high quality imaging data. On 3/9/2017 3:28 PM, Marcia Boulina wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear all, > I know this question comes up once per page, but could you share your favourite dye combination for the 5 colour fluorescent imaging? Lets say we're lazy and do not want to conjugate our AB; so which commercial dyes do you normally pick? > > I have today picked Alexa 488,555,594 and DyLight 680+DAPI. My Alexa 555 labeling will be extremely strong, so I am hoping to use a very narrow slit on an SP5. Alexa 488 and 555 will be cytoplasmic; 594 and 680 will be membrane. My considerations were: not to have membrane proteins stained in the autofluorescent range; to have the weakest staining in the cleanest channel (680). > > So do you advertise to users something more exotic on regular basis? Do you prophesy quantum dots or BV dyes? do you flip DAPI and put your proteins in the UV range? > > those will be frozen tissue sections. > Thanks in advance! > Marcia -- George McNamara, PhD Houston, TX 77054 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75/ http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 |
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In reply to this post by Marcia Boulina
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi George, I was considering a BV 421-BV510-BV605-BV711 combination or BV421+Alexa 488,561,647 However, problems are: DAPI will require extra effort (or fit a far red nuclear stain in?) using UV for excitation might be an issue for thicker tissue as opposed to cell imaging I have not looked well, but it seems like BD sells dyes or directly conjugated ab (no secondary available?) user will have to spend more bench time for AB labelling, etc Have you personally tried those dyes in multiplex experiments? we have done BV421, it looked OK for a surface marker, but not too bright (but i have not seen the same protein labelled with anything else) Are you using Q-dots at all? Thank you! M |
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