Re: Lipid Staining Help **tech support response**

[Roy Edward-2]

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Dear Anastasia,

Did you achieve a resolution to this issue?
Although I see you are using DRAQ5 (our product) I saw the quick responses
from others and thought you¹d received good sensible advice that I
couldn¹t add anything to.

However, on further reflection it may be that the combination of
lipophilic probes/dyes working together will increase the lipophilicity
generally and increase non-specificity and crosstalk between the
probes/dyes.  For example, DRAQ5, though being a DNA specific probe that
intercalates into dsDNA, is lipophilic - a necessary property for it to
behave as a cell-permeant probe.  The postulated increased lipophilicity
might then re-balance the preference for the specific target and
non-specific target accordingly.

As an alternative, I would suggest the related DNA probe DRAQ7 which is
identically far-red fluorescing and dsDNA specific but is highly watery
(it is designed as a cell-impermeant real-time viability probe) and
functions very well as a nuclear counterstain when directly compared to
DRAQ5 in fixed samples.

DRAQ7 has been cited as a counterstain for fixed cells and tissues.
One such example is:
Palacios-Reyes, et al. ³Williams' neural stem cells: new model for insight
into microRNA dysregulation."
Frontiers in Bioscience (Elite edition) 5 (2012): 1057-1073.


I hope that might help.
Don¹t hesitate to contact me off-list if you have specific questions about
this.

Kind regards,
Roy

Roy Edward
roy(at)biostatus(dot)com

BioStatus Limited
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>----------------------------------------------------------------------
>
>Date:    Thu, 17 Sep 2015 17:13:31 +0200
>From:    Anastasia Timofiiv <[hidden email]>
>Subject: Lipid Staining Help
>
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>*****
>
>Hello,
>
>  I have encountered an interesting problem and was wondering whether
>anyone else has ever seen this before:
>
>  I am using fixed (4% PFA, room temperature fixation for 20min) HeLa and
>Huh7 cells that were treated with 500=C2=B5M Oleic Acid (an omega-9 fatty
>a=
>cid)
>and am staining for lipids and cholesterol using the following dyes:
>
>1. DRAQ5 (nuclear stain)
>2. NileRed (targets lipids)
>3. BODIPY 493/503 (targets lipids)
>4. Filipin III (targets cholesterol)
>
>and am imaging using fluorescence microscopy. If I fix, stain, and image
>on
>the same day, everything looks as expected (nuclear dye stains properly,
>lipid droplets visible). If I re-image the same slides even 48h later,
>then
>major changes occur, making the slides incomparable. Firstly, the DRAQ5 no
>longer stains the nucleus, but rather becomes co-localized with the lipid
>dyes. Secondly, the lipid droplets begin to be less sharp and visible, and
>thirdly the signal to noise decreases rapidly. The cholesterol staining
>(Filipin III) works fine and is perfectly stable.
>
>I am using Mowiol as a mounting medium, and as a test during
>trouble-shooting I tried mounting using Vectashield and that made it much
>worse. The cells immediately looked as if the slide had been sitting for
>48h or so. Longer fixation (30min) did not change anything. The PFA is
>self-made (not commercial) and kept frozen in aliquots, but using
>commercial PFA had no effect.
>
>I know that PFA fixes proteins and not lipids, but it is assumed that the
>lipids become immobilized as well due to being "trapped" by fixed
>proteins.
>Others have stained for lipids and I have looked for possible explanations
>for what I'm seeing, but I can't seem to get anywhere.
>
>My solution is to simply fix, stain, and image all on the same day in
>order
>to get meaningful images, but it would be nice to know why this happens
>and
>whether this is a common problem for lipid staining.
>
>Thank you!
>Anastasia