Nile Red

[Renato Mortara]

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Shane,

If you fix/permeabilize with Tween, probably all the lipids will be
extracted. Also, 2h in PFA seems far too much. I would try 30 min, and
label with Nile Red without permeabilizing.

Best

Renato Mortara

Dr. Renato Arruda Mortara
Parasitology Division
Escola Paulista de Medicina - UNIFESP
Rua Botucatu,  862 6th floor
04023-062
São Paulo SP Brazil
Phone: [phone]
www.ecb.epm.br/~ramortara

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Dear Group,

I hope someone can help me with this problem.

I would like to stain M. tuberculosis cells with Nile Red to investigate
lipid accumulation. This
is well published, although literature seems to lack important details.

My issue is that the cells are required to remain in BSL - 3 lab and our
microscope is outside
the lab!

Could I perform the staining protocol, then fix my cells in 4 %
formaldehyde (in PBS + 0.05
% tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of
cells, thus removal
from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.

My concerns about post fixation is that it will quench or remove the dye?
Or should I first
fix, then apply Nile Red?

[Knecht, David]

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I don’t think this is true.  I seem to remember putting live cells in Tween 20 and it had no effect on the cells at all.  We always use Triton to permeabilize.  I agree on the fix.  We never go more than 10 minutes.  Dave

On Jan 13, 2014, at 11:53 AM, Renato Mortara <[hidden email]<mailto:[hidden email]>> wrote:

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Shane,

If you fix/permeabilize with Tween, probably all the lipids will be
extracted. Also, 2h in PFA seems far too much. I would try 30 min, and
label with Nile Red without permeabilizing.

Best

Renato Mortara

Dr. Renato Arruda Mortara
Parasitology Division
Escola Paulista de Medicina - UNIFESP
Rua Botucatu,  862 6th floor
04023-062
São Paulo SP Brazil
Phone: [phone]
www.ecb.epm.br/~ramortara

*****
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Dear Group,

I hope someone can help me with this problem.

I would like to stain M. tuberculosis cells with Nile Red to investigate
lipid accumulation. This
is well published, although literature seems to lack important details.

My issue is that the cells are required to remain in BSL - 3 lab and our
microscope is outside
the lab!

Could I perform the staining protocol, then fix my cells in 4 %
formaldehyde (in PBS + 0.05
% tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of
cells, thus removal
from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.

My concerns about post fixation is that it will quench or remove the dye?
Or should I first
fix, then apply Nile Red?

David Knecht, Ph.D.
Professor and Head of Core Microscopy Facility
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Shane van Breda]

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Thanks,

I was thinking I would do the fixation on poly - l - lysine slides first, then I will remove the cells from the lab followed by staining on the slide.

The protocol I have suggests making a stock in Ethanol. Do you agree?

Shane

> On 13 Jan 2014, at 18:53, Renato Mortara <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane,
>
> If you fix/permeabilize with Tween, probably all the lipids will be
> extracted. Also, 2h in PFA seems far too much. I would try 30 min, and
> label with Nile Red without permeabilizing.
>
> Best
>
> Renato Mortara
>
> Dr. Renato Arruda Mortara
> Parasitology Division
> Escola Paulista de Medicina - UNIFESP
> Rua Botucatu,  862 6th floor
> 04023-062
> São Paulo SP Brazil
> Phone: [phone]
> www.ecb.epm.br/~ramortara
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Group,
>
> I hope someone can help me with this problem.
>
> I would like to stain M. tuberculosis cells with Nile Red to investigate
> lipid accumulation. This
> is well published, although literature seems to lack important details.
>
> My issue is that the cells are required to remain in BSL - 3 lab and our
> microscope is outside
> the lab!
>
> Could I perform the staining protocol, then fix my cells in 4 %
> formaldehyde (in PBS + 0.05
> % tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of
> cells, thus removal
> from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.
>
> My concerns about post fixation is that it will quench or remove the dye?
> Or should I first
> fix, then apply Nile Red?

[Tung-Ling Chen]

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We routinely apply Nile Red to PFA fixed samples (green algae, plant tissues).  0.1% Triton slightly disrupt LD morphology, so apply nile red without permeabilization would be preferable if LD morphology is critical.  Also, applying glycerol resulted in high background fluorescence in nile red stained sample.

李冬齡Tung-Ling Li Chen, Ph.D.
Visiting Associat Fellow
Institute of Plant and Microbial Biology
Academia Sinica



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David
Sent: Tuesday, January 14, 2014 1:05 AM
To: [hidden email]
Subject: Re: Nile Red

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I don’t think this is true.  I seem to remember putting live cells in Tween 20 and it had no effect on the cells at all.  We always use Triton to permeabilize.  I agree on the fix.  We never go more than 10 minutes.  Dave

On Jan 13, 2014, at 11:53 AM, Renato Mortara <[hidden email]<mailto:[hidden email]>> wrote:

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*****

Shane,

If you fix/permeabilize with Tween, probably all the lipids will be extracted. Also, 2h in PFA seems far too much. I would try 30 min, and label with Nile Red without permeabilizing.

Best

Renato Mortara

Dr. Renato Arruda Mortara
Parasitology Division
Escola Paulista de Medicina - UNIFESP
Rua Botucatu,  862 6th floor
04023-062
São Paulo SP Brazil
Phone: [phone]
www.ecb.epm.br/~ramortara

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Group,

I hope someone can help me with this problem.

I would like to stain M. tuberculosis cells with Nile Red to investigate lipid accumulation. This is well published, although literature seems to lack important details.

My issue is that the cells are required to remain in BSL - 3 lab and our microscope is outside the lab!

Could I perform the staining protocol, then fix my cells in 4 % formaldehyde (in PBS + 0.05 % tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of cells, thus removal from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.

My concerns about post fixation is that it will quench or remove the dye?
Or should I first
fix, then apply Nile Red?

David Knecht, Ph.D.
Professor and Head of Core Microscopy Facility Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Shane van Breda]

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*****

I will prepare cells with and without Tween 80 and see what happens. Now that I can't use glycerol as a mounting media, what else would you suggest? Standard 1 x PBS? I am not too worried about slide storage.

Regards,

Shane

> On 14 Jan 2014, at 3:08, Tung-Ling Chen <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We routinely apply Nile Red to PFA fixed samples (green algae, plant tissues).  0.1% Triton slightly disrupt LD morphology, so apply nile red without permeabilization would be preferable if LD morphology is critical.  Also, applying glycerol resulted in high background fluorescence in nile red stained sample.
>
> 李冬齡Tung-Ling Li Chen, Ph.D.
> Visiting Associat Fellow
> Institute of Plant and Microbial Biology
> Academia Sinica
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David
> Sent: Tuesday, January 14, 2014 1:05 AM
> To: [hidden email]
> Subject: Re: Nile Red
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I don’t think this is true.  I seem to remember putting live cells in Tween 20 and it had no effect on the cells at all.  We always use Triton to permeabilize.  I agree on the fix.  We never go more than 10 minutes.  Dave
>
> On Jan 13, 2014, at 11:53 AM, Renato Mortara <[hidden email]<mailto:[hidden email]>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane,
>
> If you fix/permeabilize with Tween, probably all the lipids will be extracted. Also, 2h in PFA seems far too much. I would try 30 min, and label with Nile Red without permeabilizing.
>
> Best
>
> Renato Mortara
>
> Dr. Renato Arruda Mortara
> Parasitology Division
> Escola Paulista de Medicina - UNIFESP
> Rua Botucatu,  862 6th floor
> 04023-062
> São Paulo SP Brazil
> Phone: [phone]
> www.ecb.epm.br/~ramortara
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Group,
>
> I hope someone can help me with this problem.
>
> I would like to stain M. tuberculosis cells with Nile Red to investigate lipid accumulation. This is well published, although literature seems to lack important details.
>
> My issue is that the cells are required to remain in BSL - 3 lab and our microscope is outside the lab!
>
> Could I perform the staining protocol, then fix my cells in 4 % formaldehyde (in PBS + 0.05 % tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of cells, thus removal from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.
>
> My concerns about post fixation is that it will quench or remove the dye?
> Or should I first
> fix, then apply Nile Red?
>
> David Knecht, Ph.D.
> Professor and Head of Core Microscopy Facility Department of Molecular and Cell Biology
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)

[Tung-Ling Chen]

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*****

I generally freeze the slides in -20oC with fixed cells and stain with Nile Red right before viewing.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shane van Breda
Sent: Tuesday, January 14, 2014 11:45 AM
To: [hidden email]
Subject: Re: Nile Red

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I will prepare cells with and without Tween 80 and see what happens. Now that I can't use glycerol as a mounting media, what else would you suggest? Standard 1 x PBS? I am not too worried about slide storage.

Regards,

Shane

> On 14 Jan 2014, at 3:08, Tung-Ling Chen <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We routinely apply Nile Red to PFA fixed samples (green algae, plant tissues).  0.1% Triton slightly disrupt LD morphology, so apply nile red without permeabilization would be preferable if LD morphology is critical.  Also, applying glycerol resulted in high background fluorescence in nile red stained sample.
>
> 李冬齡Tung-Ling Li Chen, Ph.D.
> Visiting Associat Fellow
> Institute of Plant and Microbial Biology Academia Sinica
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Knecht, David
> Sent: Tuesday, January 14, 2014 1:05 AM
> To: [hidden email]
> Subject: Re: Nile Red
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I don’t think this is true.  I seem to remember putting live cells in
> Tween 20 and it had no effect on the cells at all.  We always use
> Triton to permeabilize.  I agree on the fix.  We never go more than 10
> minutes.  Dave
>
> On Jan 13, 2014, at 11:53 AM, Renato Mortara <[hidden email]<mailto:[hidden email]>> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Shane,
>
> If you fix/permeabilize with Tween, probably all the lipids will be extracted. Also, 2h in PFA seems far too much. I would try 30 min, and label with Nile Red without permeabilizing.
>
> Best
>
> Renato Mortara
>
> Dr. Renato Arruda Mortara
> Parasitology Division
> Escola Paulista de Medicina - UNIFESP
> Rua Botucatu,  862 6th floor
> 04023-062
> São Paulo SP Brazil
> Phone: [phone]
> www.ecb.epm.br/~ramortara
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Group,
>
> I hope someone can help me with this problem.
>
> I would like to stain M. tuberculosis cells with Nile Red to investigate lipid accumulation. This is well published, although literature seems to lack important details.
>
> My issue is that the cells are required to remain in BSL - 3 lab and our microscope is outside the lab!
>
> Could I perform the staining protocol, then fix my cells in 4 % formaldehyde (in PBS + 0.05 % tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of cells, thus removal from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.
>
> My concerns about post fixation is that it will quench or remove the dye?
> Or should I first
> fix, then apply Nile Red?
>
> David Knecht, Ph.D.
> Professor and Head of Core Microscopy Facility Department of Molecular
> and Cell Biology
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)

[Stanislav Vitha-2]

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Do you know if the chosen fixation protocol will actually kill the cells so that
they can be taken out of the BSL3 facility?   Apart from doing an actual
viability test by plating the fixed cells on agar medium, are there some
established protocols for these situations?  I am asking because every now
and then I am in a similar situation, asked to do imaging on samples that
are above our BSL permit.

Stan Vitha
Microscopy and Imaging Center
Texas A&M University


On Mon, 13 Jan 2014 14:53:37 -0200, Renato Mortara
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Shane,
>
>If you fix/permeabilize with Tween, probably all the lipids will be
>extracted. Also, 2h in PFA seems far too much. I would try 30 min, and
>label with Nile Red without permeabilizing.
>
>Best
>
>Renato Mortara
>
>Dr. Renato Arruda Mortara
>Parasitology Division
>Escola Paulista de Medicina - UNIFESP
>Rua Botucatu,  862 6th floor
>04023-062
>São Paulo SP Brazil
>Phone: [phone]
>www.ecb.epm.br/~ramortara
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Group,
>
>I hope someone can help me with this problem.
>
>I would like to stain M. tuberculosis cells with Nile Red to investigate
>lipid accumulation. This
>is well published, although literature seems to lack important details.
>
>My issue is that the cells are required to remain in BSL - 3 lab and our
>microscope is outside
>the lab!
>
>Could I perform the staining protocol, then fix my cells in 4 %
>formaldehyde (in PBS + 0.05
>% tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of
>cells, thus removal
>from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.
>
>My concerns about post fixation is that it will quench or remove the dye?
>Or should I first
>fix, then apply Nile Red?

[Shane van Breda]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have done the viability testing and it checks out.

I also know you can apply KMnO4 for about one minute, that will kill the cells.

Regards,

Shane

> On 14 Jan 2014, at 16:49, Stanislav Vitha <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Do you know if the chosen fixation protocol will actually kill the cells so that
> they can be taken out of the BSL3 facility?   Apart from doing an actual
> viability test by plating the fixed cells on agar medium, are there some
> established protocols for these situations?  I am asking because every now
> and then I am in a similar situation, asked to do imaging on samples that
> are above our BSL permit.
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
> On Mon, 13 Jan 2014 14:53:37 -0200, Renato Mortara
> <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Shane,
>>
>> If you fix/permeabilize with Tween, probably all the lipids will be
>> extracted. Also, 2h in PFA seems far too much. I would try 30 min, and
>> label with Nile Red without permeabilizing.
>>
>> Best
>>
>> Renato Mortara
>>
>> Dr. Renato Arruda Mortara
>> Parasitology Division
>> Escola Paulista de Medicina - UNIFESP
>> Rua Botucatu,  862 6th floor
>> 04023-062
>> São Paulo SP Brazil
>> Phone: [phone]
>> www.ecb.epm.br/~ramortara
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Group,
>>
>> I hope someone can help me with this problem.
>>
>> I would like to stain M. tuberculosis cells with Nile Red to investigate
>> lipid accumulation. This
>> is well published, although literature seems to lack important details.
>>
>> My issue is that the cells are required to remain in BSL - 3 lab and our
>> microscope is outside
>> the lab!
>>
>> Could I perform the staining protocol, then fix my cells in 4 %
>> formaldehyde (in PBS + 0.05
>> % tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of
>> cells, thus removal
>> from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.
>>
>> My concerns about post fixation is that it will quench or remove the dye?
>> Or should I first
>> fix, then apply Nile Red?