fixable, dead cell nuclear counterstain.

[daj1u06]

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Can anyone recommend a non membrane permiable, fixable, nuclear counterstain, which neither fluoresces far red, nor requires hard UV excitation (so DAPI is out) which can then withstand wax embedding for histology? We have a user who wants to test a novel delivery system into which an aqueous phase dye can be incorporated, the test being that if the delivery system gets into a cell and is processed, the dye will be released and enter the nucleus to bind the DNA and stain the cell. The experiments will be in vivo, hence there is a need to fix and wax embed.

I know that there are membrane impermeant, fixable cytosolic amine reactive dyes which would stain the cell if delivered this way but wondered about a nuclear stain.

There seems relatively little literature on fixable DNA dyes or does one assume that aldehyde based fixation will cross-link any already incorporated dye.

Thanks in advance,

Dave Johnston,
Biomedical Imaging Unit,
Southampton.

[George McNamara]

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PI = Propidium Iodide. Excitation max ~540 nm (excites ok at 488 nm),
emission max ~618 nm. Spectra on many spectral websites, for example
https://searchlight.semrock.com  and data in PubSpectra XLSX file at
https://works.bepress.com/gmcnamara/9


I suggest your user compare their novel delivery system to SLO, as
published by Teng ... Belmont, Selvin 2016 -- which could be a good role
model of experiments to do for publication ... and ELife could be a good
place to send the manuscript.

https://www.ncbi.nlm.nih.gov/pubmed/?term=belmont+selvin

Elife. 2016 Dec 9;5. pii: e20378. doi: 10.7554/eLife.20378.
Labeling proteins inside living cells using external fluorophores for
microscopy.
Teng KW1,2, Ishitsuka Y2,3, Ren P1,2, Youn Y1,2, Deng X4, Ge P3, Belmont
AS1,4, Selvin PR1,2,3,4.

Abstract
Site-specific fluorescent labeling of proteins inside live mammalian
cells has been achieved by employing Streptolysin O, a bacterial enzyme
which forms temporary pores in the membrane and allows delivery of
virtually any fluorescent probes, ranging from labeled IgG's to small
ligands, with high efficiency (>85% of cells). The whole process,
including recovery, takes 30 min, and the cell is ready to be imaged
immediately. A variety of cell viability tests were performed after
treatment with SLO to ensure that the cells have intact membranes, are
able to divide, respond normally to signaling molecules, and maintains
healthy organelle morphology. When combined with Oxyrase, a
cell-friendly photostabilizer, a ~20x improvement in fluorescence
photostability is achieved. By adding in glutathione, fluorophores are
made to blink, enabling super-resolution fluorescence with 20-30 nm
resolution over a long time (~30 min) under continuous illumination.
Example applications in conventional and super-resolution imaging of
native and transfected cells include p65 signal transduction activation,
single molecule tracking of kinesin, and specific labeling of a series
of nuclear and cytoplasmic protein complexes.
KEYWORDS:
Fluorescence; HaloTag; biophysics; cell biology; human; live-cell
imaging; mouse; nanobody; single molecule; structural biology;
super-resolution imaging
PMID: 27935478 PMCID: PMC5148600 DOI: 10.7554/eLife.20378


On 2/1/2017 8:25 AM, Dave Johnston 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.
> *****
>
> Can anyone recommend a non membrane permiable, fixable, nuclear counterstain, which neither fluoresces far red, nor requires hard UV excitation (so DAPI is out) which can then withstand wax embedding for histology? We have a user who wants to test a novel delivery system into which an aqueous phase dye can be incorporated, the test being that if the delivery system gets into a cell and is processed, the dye will be released and enter the nucleus to bind the DNA and stain the cell. The experiments will be in vivo, hence there is a need to fix and wax embed.
>
> I know that there are membrane impermeant, fixable cytosolic amine reactive dyes which would stain the cell if delivered this way but wondered about a nuclear stain.
>
> There seems relatively little literature on fixable DNA dyes or does one assume that aldehyde based fixation will cross-link any already incorporated dye.
>
> Thanks in advance,
>
> Dave Johnston,
> Biomedical Imaging Unit,
> Southampton.

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Michael Giacomelli]

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All dyes I have used are greatly attenuated by xylene processing,
however some remain reasonably bright.  Propidium iodine is a good
choice, although unless you deliver a reasonable amount it may be
quite dim post processing.

Mike

On Wed, Feb 1, 2017 at 9:25 AM, Dave Johnston <[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.
> *****
>
> Can anyone recommend a non membrane permiable, fixable, nuclear counterstain, which neither fluoresces far red, nor requires hard UV excitation (so DAPI is out) which can then withstand wax embedding for histology? We have a user who wants to test a novel delivery system into which an aqueous phase dye can be incorporated, the test being that if the delivery system gets into a cell and is processed, the dye will be released and enter the nucleus to bind the DNA and stain the cell. The experiments will be in vivo, hence there is a need to fix and wax embed.
>
> I know that there are membrane impermeant, fixable cytosolic amine reactive dyes which would stain the cell if delivered this way but wondered about a nuclear stain.
>
> There seems relatively little literature on fixable DNA dyes or does one assume that aldehyde based fixation will cross-link any already incorporated dye.
>
> Thanks in advance,
>
> Dave Johnston,
> Biomedical Imaging Unit,
> Southampton.

[Glen MacDonald-2]

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Dont’ know if they are aldehyde fixable, but the monomeric cyanine fluorophores from Molecular Probes survive dehydration and epoxy embedding.  they are generally membrane impermeant, but will pass through some cationic channels such as certain purinergic and mechanotransduction channels.  
Let us know what works.  

Regards,
Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

> On Feb 1, 2017, at 6:25 AM, Dave Johnston <[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.
> *****
>
> Can anyone recommend a non membrane permiable, fixable, nuclear counterstain, which neither fluoresces far red, nor requires hard UV excitation (so DAPI is out) which can then withstand wax embedding for histology? We have a user who wants to test a novel delivery system into which an aqueous phase dye can be incorporated, the test being that if the delivery system gets into a cell and is processed, the dye will be released and enter the nucleus to bind the DNA and stain the cell. The experiments will be in vivo, hence there is a need to fix and wax embed.
>
> I know that there are membrane impermeant, fixable cytosolic amine reactive dyes which would stain the cell if delivered this way but wondered about a nuclear stain.
>
> There seems relatively little literature on fixable DNA dyes or does one assume that aldehyde based fixation will cross-link any already incorporated dye.
>
> Thanks in advance,
>
> Dave Johnston,
> Biomedical Imaging Unit,
> Southampton.

[Jurkevic, Aleksandr]

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Dave,

You can try PicoGreen. It is very bright and remains bright after fixation, however it also stains mitochondrial DNA. I do not know if it can survive paraffin embedding and dewaxing.

Alexander


Alexander Jurkevich, PhD
Associate Director
Molecular Cytology Core
University of Missouri


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dave Johnston
Sent: Wednesday, February 01, 2017 8:25 AM
To: [hidden email]
Subject: fixable, dead cell nuclear counterstain.

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Can anyone recommend a non membrane permiable, fixable, nuclear counterstain, which neither fluoresces far red, nor requires hard UV excitation (so DAPI is out) which can then withstand wax embedding for histology? We have a user who wants to test a novel delivery system into which an aqueous phase dye can be incorporated, the test being that if the delivery system gets into a cell and is processed, the dye will be released and enter the nucleus to bind the DNA and stain the cell. The experiments will be in vivo, hence there is a need to fix and wax embed.

I know that there are membrane impermeant, fixable cytosolic amine reactive dyes which would stain the cell if delivered this way but wondered about a nuclear stain.

There seems relatively little literature on fixable DNA dyes or does one assume that aldehyde based fixation will cross-link any already incorporated dye.

Thanks in advance,

Dave Johnston,
Biomedical Imaging Unit,
Southampton.