Re: fixable, dead cell nuclear counterstain. **vendor response**

[Kilgore, Jason A.]

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** Vendor Response **

In order to be truly fixable (as in cross-linkable with aldehydes), they would have to either bind covalently or have free amine groups.  I don't know of any nucleic acid stains that truly fit this bill.  But.....

1) some dyes seem to be *retained* after fixation for a short while, but we did a study here at Molecular Probes of many of these dyes, via flow cytometry, to examine retention after fixation, and all of the nucleic acid binding dyes came off over times, though at somewhat different rates.  Here is a poster we generated with that data:  https://tools.thermofisher.com/content/sfs/posters/2009-dead.pdf 

2) ethidium monoazide bromide (EMA) can be crosslinked in place with bright, visible wavelength light (not aldehyde chemical crosslinking).  For imaging, it has peak ex/em at 510/600, and it should be non-membrane permeant (as with other ethidium dyes).  Paper reference:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC342796/ 

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, then option 2.
Or dial direct at [phone]
[hidden email]
www.lifetechnologies.com

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dave Johnston
Sent: Wednesday, February 01, 2017 6:25 AM
To: [hidden email]
Subject: fixable, dead cell nuclear counterstain.

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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.

[Glen MacDonald-2]

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There are some other types of cross-linking fixatives.  1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-links amino groups to carboxyl groups.  I tried it several years ago to immobilize Alexa 594 iontophoresed into patch-clamped neurons, I think our results got used in a poster, but never published.  
EDC appeared to immobilize Alexa594 for at least 2 weeks, then the dye began diffusing.  

Blended fixatives of 104 mM EDC in PBS + 4% paraformaldehyde or 52 mM EDC + 2%  paraformaldehyde were created  just before use.
400 um avian brainstem slices were incubated overnight at 4 degrees then washed in PBS.  

EDC refs: Thomas, J.O., Sternberg, N., and Weisberg, R. (1978), J Mol. Biol; 123(2):149-161,  Altered Arrangement of the DNA in Injection-defective Lambda Barcteriophage.

Willingham, M.C., Yamada, S.S., Pastan, I, (1978), Proc. Nat. Acad. Sci. USA, 75(9):4359-4363, Ultrastructural antibody localization of alpha2-macroglobulin in membrane-limited vesicles in cultured cells.
Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

> On Feb 7, 2017, at 2:46 PM, Kilgore, Jason A. <[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.
> *****
>
> ** Vendor Response **
>
> In order to be truly fixable (as in cross-linkable with aldehydes), they would have to either bind covalently or have free amine groups.  I don't know of any nucleic acid stains that truly fit this bill.  But.....
>
> 1) some dyes seem to be *retained* after fixation for a short while, but we did a study here at Molecular Probes of many of these dyes, via flow cytometry, to examine retention after fixation, and all of the nucleic acid binding dyes came off over times, though at somewhat different rates.  Here is a poster we generated with that data:  https://tools.thermofisher.com/content/sfs/posters/2009-dead.pdf 
>
> 2) ethidium monoazide bromide (EMA) can be crosslinked in place with bright, visible wavelength light (not aldehyde chemical crosslinking).  For imaging, it has peak ex/em at 510/600, and it should be non-membrane permeant (as with other ethidium dyes).  Paper reference:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC342796/ 
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Life Sciences Solutions
>
> Thermo Fisher Scientific
> 29851 Willow Creek Rd.
> Eugene, OR  97402-9132
> 1-800-955-6288 then option 4, then option 6, then option 2.
> Or dial direct at [phone]
> [hidden email]
> www.lifetechnologies.com
>
> This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dave Johnston
> Sent: Wednesday, February 01, 2017 6:25 AM
> To: [hidden email]
> Subject: fixable, dead cell nuclear counterstain.
>
> *****
> 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.