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---- Original message ----
>Date: Fri, 16 Dec 2005 11:08:05 +0100
>From: Ingela Parmryd <[log in to unmask]>
>Subject: SV: antifade
>To: [log in to unmask]
>
> Search the CONFOCAL archive at
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> Unless staining only a subpopulation of your
> antigen is not a problem in your experiments, I
> would not recommend high dilution of a primary
> antibody just because an antifade reagent allows you
> to detect the signal. If you for instance are
> studying colocalisation you then risk ending up with
> an incorrect result.
>
> Ingela Parmryd
>
> -----Ursprungligt meddelande-----
> Fraan: Confocal Microscopy List
> [mailto:[log in to unmask]]Fo:r Michael
> Mancini
> Skickat: 15 December 2005 23:26
> Till: [log in to unmask]
> A:mne: antifade
>
> Search the CONFOCAL archive at
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> Speaking of new antifade solutions, I have to add
> a comment on the newer MolProbes "gold" series.
> I was skeptical at first ("Oh, another product to
> try?") but I'm pleased to say they really do
> result in MUCH brighter signals over their
> original formulation. Even on super bright
> samples, we are routinely seeing several fold
> increase in signal with the Gold Series (both
> Prolong and Slowfade). My guess is that with some
> low level signals it will be an even larger
> difference. I expect that in the end we'll save
> money on primary antibodies as we can further
> dilute them and still see a strong signal. On the
> other hand, we did try the DAPI containing
> formulations of Prolong/Slowfade to see if we
> could skip our usual DAPI stain. The DAPI signal
> obtained from the mounting media is fine, but
> about 2x lower than what I'm used to. Maybe we
> just like screaming nuclei more than the next
> guy....
> Mike Mancini
> Baylor College of Medicine
> (and, no, I don't own shares in Invitrogen,
> etc...!)
> On Dec 15, 2005, at 4:06 PM, Ignatius, Mike wrote:
>
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S1=confocal
> Caution Vendor Response. Comments on bubbles
> from anti-oxidants in antifades, dye brightness
> and additional mounting media issues.
> At Molecular Probes we noticed bubbles with some
> vendors products, although more in the hardening
> versions. We sell a number of antifade mounting
> media with anti-oxidants in them. No one has
> reported bubbles in our Prolong Gold antifade
> mounting media, or our original Prolong or
> Slowfade antifade. So antioxidants alone can't
> be producing the problem - we suspect the
> hardener generates these bubbles.
> Our new versions, Prolong Gold and Slowfade Gold
> stabilizes or increases initial brightness for
> all dyes from blue to IR. The biggest gains are
> seen in brightness and photostability of red and
> NIR dyes, where some users have described
> greater than 10x improvements. We strongly agree
> that many mounting media do decrease initial
> brightness, including the many home brew
> formulations. This decrease is often never
> observed by users, since the first look at a
> prep is often after fully processing the sample.
> The RI's of Prolong and Prolong Gold is matched
> to oil. We recommend the redesigned glycerol
> version of Slowfade, now SlowFade Gold, for
> thick specimens (e.g. confocal 3D work) where
> the the bulk of the material is closer to 1.3.
> Both products are now one step solutions: no
> mixing or centrifugation. However, if bleaching
> experiments are being done we recommend our
> original Prolong mounting media product, leaving
> out the second vial of anti-oxidant.
> As an aside, Prolong Gold does not work with
> semi-conductor based Quantum Dots, a 90% a
> glycerol/PBS is currently recommended.
> If interested, a more detailed technological
> characterization can be forwarded to
> individuals.
> Mike
> Mike Ignatius, PhD
> Director of Cell Biology and Imaging
> Invitrogen/Molecular Probes
> 29851 Willow Creek Road
> Eugene, Oregon 97402
> 541 335-0414 office
> 541 510-1736 cell
> [log in to unmask]
>
> ------------------------------------------------
>
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf
> Of carl
> Sent: Wednesday, December 14, 2005 1:23 PM
> To: [log in to unmask]
> Subject: bubbles
> Search the CONFOCAL archive at
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S1=confocal
> Hello all,
> A confocal user is getting some results that I
> am unable to rationalize. Neurons cultured in
> dishes with coverslip bottoms are fixed and
> stained with Mitotracker red, then Vectashield
> is used as a mounting medium. Occasionally a
> bubble occurs between the two coverslips, and
> the conundrum comes from the different image
> quality of the cells in the bubble vs. those in
> the mounting medium. The staining seems
> brighter, more distinct, with less background
> for the cells in the bubble than the cells in
> mounting medium. Because the dish is viewed
> upside down with our upright microscope, the
> bubble is on the other side of the cells
> relative to the excitation and emission light.
> Should we not be using an antifade, any mounting
> medium, or is this specifically a Vectashield
> thing?
> Any thoughts?
> Thanks very much,
> Carl
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-626-8469
> FAX 520-621-3709
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