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Hi Shane,
Probably going to be more (secondary) antibody and lot dependent (and
possibly company), rather than fluorophore per se. Either will be far
superior to fluorescein if the pH of the mounting medium is less than ~7.5.
Leica's STED preparation guide recommends DyLight 488 over Alexa Fluor
488. At a Leica CW-STED demo I brought user slides to, we obtained
excellent super-resolution results with several lab's Alexa Fluor 488
conjugated secondary antibody slides. We also had several Alexa Fluor
488 slides that had no resolution advantage to confocal only, and one
DyLight 488 slide with no advantage - I believe (but cannot prove with
limited specimens) that "no improvement" specimens were refractive index
mismatch, specimen distance from coverglass, or mounting media not being
perfect (seer Kasper .. Hell 2010 Small for what an optimized ROXS
mounting medium can do for STED). We also obtained excellent results
with a CYTOO-prepared micropatterned coverslip that had
fluorescein-phalloidin and amazing super-resolution GFP-fusion protein
live /E coli/ cells: EGFP did not photobleach at all, even with
prolonged 488 excitation and full power, ~~2 W (at laser) 594 depletion
illumination! Alexa Fluor 488 and fluorescein photobleached fairly
quickly in STED mode on most of the specimens, and various red
fluorphores bleached impressively well with the 592 nm depletion beam
on. I am extremely interested in whether researchers have found Leica's
Ti:Sapphire depletion STED to be more useful than 592 nm CW-STED - any
experiences on the listserv or privately are welcome!
Sincerely,
George
p.s.
With respect to Cytoo, see Schauer et al 2010 Nature Methods:
Schauer K, Duong T, Bleakley K, Bardin S, Bornens M, Goud B.
Probabilistic density maps to study global endomembrane organization.
</pubmed/20512144> Nat Methods. 2010 Jul;7(7):560-6. Epub 2010 May
30.PMID: 20512144
With respect to indirect immunofluorescence, Richard Burry recently a
book - Immunocytochemistry - in which he showed that more washes (7+)
after each antibody incubation is better than fewer. Also critical to
make sure the cells/tissue do not have a chance to dry out during any
step. That is, if processing several slides in parallel, remove and
immediately replace wash solution from one coverglass, rather than
remove from all ten then add sequentially.
My thanks to Leica, Charles Hemphill (Southeast USA Leica confocal/STED
sales) and Max Planck Florida Institute for enabling me to get time on
the CW-STED nanoscope.
On 12/11/2010 9:55 AM, Shane van Breda wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear members,
>
> I was wondering what you guys might suggest is a better antibody conjugate
> to use for confocal microscopy?
>
> DyLight or Alexa Fluor?
>
> Them seem very similar, so do I choose either of them?
>
> Thanks,
>
> Shane.
>
>
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