Lipid Staining Help

[Anastasia Timofiiv]

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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 500µM Oleic Acid (an omega-9 fatty acid)
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

[Sylvie Le Guyader]

Hi Anastasia

We have the same problem with the LipidTox dye and PBS on the cells.
We stain and image immediately.
However I think this doesn’t happen with Bodipy dyes.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Unit Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Anastasia Timofiiv
Sent: den 17 september 2015 17:14
To: [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 500µM Oleic Acid (an omega-9 fatty acid) 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

[Glen MacDonald-2]

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dear Anastasia,
Draq-5 and other far red fluorophores tend to be lipophilic and will move into lipid domains.  However,I would have thought that Draq-5 would be too tightly bound.  Perhaps try a lower concentration.
We had a similar problem with the lipophilic membrane labels DiI, DiO, etc. diffusing from the membrane lipid layer when they were mounted in glycerol, Vectashield and some other mountants.  that problem was largely overcome by mounting with 80% glycerol+20% phosphate buffer to limit dye solubility.

  Regards,
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Sep 17, 2015, at 8:13 AM, Anastasia Timofiiv <[hidden email]> 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.
> *****
>
> 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 500µM Oleic Acid (an omega-9 fatty acid)
> 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

[Mel Symeonides]

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*****

These lipid dyes will just leak out of the membranes over time, as you
experienced, unless they are chloromethylated so they can undergo
aldehyde fixation. We used the fixable version of Vybrant DiI, Vybrant
CM-DiI, to label our cells when we needed to fix for imaging. There are
no other CM versions of the Vybrant dyes, as far as I'm aware, but maybe
you can push Molecular Probes to start making some.

Another alternative you could try is fluorophore-conjugated wheat germ
agglutinin. This will stain the plasma membrane nicely in many cell types.

Be aware that in case you are permeabilizing for intracellular staining,
most detergents will wash out lipid dyes, so use a gentler, more
specific detergent, such as digitonin (though if you are also doing
cholesterol staining, digitonin might wash out those dyes - I haven't
tried that specifically, I just know digitonin plays nice with Vybrant
CM-DiI).

Regards,
Mel

On 9/17/2015 12:19 PM, Glen MacDonald 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 Anastasia,
> Draq-5 and other far red fluorophores tend to be lipophilic and will move into lipid domains.  However,I would have thought that Draq-5 would be too tightly bound.  Perhaps try a lower concentration.
> We had a similar problem with the lipophilic membrane labels DiI, DiO, etc. diffusing from the membrane lipid layer when they were mounted in glycerol, Vectashield and some other mountants.  that problem was largely overcome by mounting with 80% glycerol+20% phosphate buffer to limit dye solubility.
>
>    Regards,
> Glen MacDonald
> Core for Communication Research
> Virginia Merrill Bloedel Hearing Research Center
> Cellular Morphology Core
> Center on Human Development and Disability
> Box 357923
> University of Washington
> Seattle, WA 98195-7923  USA
> [phone]
> [hidden email]
>
>
>
>
>
>
>
> On Sep 17, 2015, at 8:13 AM, Anastasia Timofiiv <[hidden email]> 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.
>> *****
>>
>> 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 500µM Oleic Acid (an omega-9 fatty acid)
>> 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

--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]


--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]

[Jason Miller]

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*****

We haven't had the problem with BODIPY dyes, but we've also found that LD are way easier to quantify, and are much more stable over time for imaging, using ADRP antibodies. You have to be quite gentle with your permeabilazation - small amounts of digitonin for short periods of time - but it works great. Contact me off-line if you want more details.

-Jason


-------

Jason Miller, MD, PhD

University of Michigan Kellogg Eye Center



Home address:

117 Worden Ave

Ann Arbor, MI 48103

Cell: [phone]

E-mail: [hidden email]<mailto:[hidden email]>



"When I was 5 years old, my mother always told me that happiness was the key to life. When I went to school, they asked me what I wanted to be when I grew up. I wrote down, 'Happy.' They told me I didn't understand the assignment. I told them they didn't understand life." - John Lennon

On Thu, Sep 17, 2015 at 11:51 AM, Sylvie Le Guyader <[hidden email]<mailto:[hidden email]>> wrote:
Hi Anastasia

We have the same problem with the LipidTox dye and PBS on the cells.
We stain and image immediately.
However I think this doesn't happen with Bodipy dyes.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Unit Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]<tel:%2B46%20%280%29%2073%20733%205008>
office: [phone]<tel:%2B46%20%280%29%208%205248%201107>
LCI website

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]<mailto:[hidden email]>] On
Behalf Of Anastasia Timofiiv
Sent: den 17 september 2015 17:14
To: [hidden email]<mailto:[hidden email]>
Subject: Lipid Staining Help

*****
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<http://www.imgur.com/> and include the link in your posting.
*****

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 500µM Oleic Acid (an omega-9 fatty acid)
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

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