Nuclear stains and Tissue Clearing

[Donald Alexander Fowler]

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Dear Confocal Microscopy,

Does anybody know of a good nuclear stain that will stay specific to the
nuclei when cleared in BABB or Dibenzyl ether?  I've tried in embryonic
chick tissue and have had very poor results.

Kind Regards,

Donald

--
Donald Alexander Fowler
PhD Candidate
Department of Biology
McGill University

[Glen MacDonald-2]

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the monomeric cyanines To-pro-3, yo-pro-1 for post-fix staining.  Haven’t tried Draq-5 or LDS 751.


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 Jul 10, 2014, at 10:33 AM, Donald Alexander Fowler <[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.
> *****
>
> Dear Confocal Microscopy,
>
> Does anybody know of a good nuclear stain that will stay specific to the
> nuclei when cleared in BABB or Dibenzyl ether?  I've tried in embryonic
> chick tissue and have had very poor results.
>
> Kind Regards,
>
> Donald
>
> --
> Donald Alexander Fowler
> PhD Candidate
> Department of Biology
> McGill University

[Craig Brideau]

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Shot in the dark here, but Propidium Iodide possibly?
I am not sure how well it will hold up to the clearing, but in my
experience it is a fairly strong fluorophore, and it isn't very expensive
so it might be worth a try.

Craig


On Thu, Jul 10, 2014 at 11:33 AM, Donald Alexander Fowler <
[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.
> *****
>
> Dear Confocal Microscopy,
>
> Does anybody know of a good nuclear stain that will stay specific to the
> nuclei when cleared in BABB or Dibenzyl ether?  I've tried in embryonic
> chick tissue and have had very poor results.
>
> Kind Regards,
>
> Donald
>
> --
> Donald Alexander Fowler
> PhD Candidate
> Department of Biology
> McGill University
>

[Andrea Stout]

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Donald,

Another vote for propidium iodide here.  Cleared brain tissue prepared by one of our core’s customers (CLARITY) looked great under conventional confocal imaging conditions. Very bright through the entire sample (which was over 3 mm thick). We were able to image nuclei through a depth of over 700 microns (10x, 0.4 NA, 488 nm excitation) and could have gone deeper if necessary.

Andrea L. Stout, Ph. D.
Director,  CDB Microscopy Core Facility
Perelman School of Medicine at the University of Pennsylvania


On Jul 10, 2014, at 3:32 PM, Craig Brideau <[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.
> *****
>
> Shot in the dark here, but Propidium Iodide possibly?
> I am not sure how well it will hold up to the clearing, but in my
> experience it is a fairly strong fluorophore, and it isn't very expensive
> so it might be worth a try.
>
> Craig
>
>
> On Thu, Jul 10, 2014 at 11:33 AM, Donald Alexander Fowler <
> [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.
>> *****
>>
>> Dear Confocal Microscopy,
>>
>> Does anybody know of a good nuclear stain that will stay specific to the
>> nuclei when cleared in BABB or Dibenzyl ether?  I've tried in embryonic
>> chick tissue and have had very poor results.
>>
>> Kind Regards,
>>
>> Donald
>>
>> --
>> Donald Alexander Fowler
>> PhD Candidate
>> Department of Biology
>> McGill University
>>

[Doube, Michael]

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Hi Donald

I saw a few responses indicating propidium iodide might work: it's certainly worth a go as a bright and cheap-ish nucleic acid stain, only downside you might hit (because you say you want nuclear specificity) is that it's not completely selective for nuclear DNA and you may get staining of RNA as well. The usual approach to deal with that is an RNAse treatment, though not sure how that will work in very thick sections, or those already in a clearing agent.

Michael


On 10/07/14 18:45, Donald Alexander Fowler 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 Confocal Microscopy,
>
> Does anybody know of a good nuclear stain that will stay specific to the
> nuclei when cleared in BABB or Dibenzyl ether?  I've tried in embryonic
> chick tissue and have had very poor results.
>
> Kind Regards,
>
> Donald
>

<http://www.rvc.ac.uk>

This message, together with any attachments, is intended for the stated addressee(s) only and may contain privileged or confidential information. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Royal Veterinary College.

[Yves LUTZ]

Hi Donald,

Have you tried Q nuclear deep red stain from Life Tech designed in the QDot field (ref Q10363)?  It needs a dehydrated sample as for clearing with BABB or DBE (although perhaps as well a  special mounting medium called Q mount, ref Q 10336).

I my clearing experiments with BABB or DBE, the use of Hoechst 33342 (10-50 ug/ml) was not too bad.

To mention that non dehydrating clearing procedures work well with Hoechst ( e.g.SeeDB).

Best regards,

Yves


Dr Yves Lutz PhD
Imaging Centre - IGBMC
1, rue Laurent Fries
67404 Illkirch
FRANCE






-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Donald Alexander Fowler
Envoyé : jeudi 10 juillet 2014 19:34
À : [hidden email]
Objet : Nuclear stains and Tissue Clearing

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*****

Dear Confocal Microscopy,

Does anybody know of a good nuclear stain that will stay specific to the nuclei when cleared in BABB or Dibenzyl ether?  I've tried in embryonic chick tissue and have had very poor results.

Kind Regards,

Donald

--
Donald Alexander Fowler
PhD Candidate
Department of Biology
McGill University