recommendation for a "blue" secondary fluorochrome

[Cromey, Douglas W - (dcromey)]

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Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.

I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.

The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?

Thanks,
Doug

------------------------------------------------------------------------------------------
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

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[Alfonso Schmidt]

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Hi Douglas:

We use BV421 ( Biolegend or BD) or AF405, both works very good in our confocal ( FV1200) exciting with laser 405 and collecting emission with a BD 430-450.


Alfonso Schmidt
Hugh Green Cytometry Core
Staff Scientist

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> On Aug 21, 2018, at 11:06 AM, Cromey, Douglas W - (dcromey) <[hidden email]> wrote:
>
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> *****
>
> Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.
>
> I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.
>
> The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?
>
> Thanks,
> Doug
>
> ------------------------------------------------------------------------------------------
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  LSN 463              email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www
> Home of: "Microscopy and Imaging Resources on the WWW"
>
> UA Microscopy Alliance -  http://microscopy.arizona.edu<http://microscopy.arizona.edu/>

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[Moulding, Dale]

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Hi Doug,


I have a user that has dropped Dapi and swears by Alexa405. Used on an LSM880 it gives great results, even in wholemounts in PBS (no antifade). I didn't believe it until I watched a few scans. Best used for your more abundant antigens but works surprisingly well.


good luck


Dale


http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/confocal-microscopy


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
Sent: 21 August 2018 00:06:28
To: [hidden email]
Subject: recommendation for a "blue" secondary fluorochrome

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Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.

I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.

The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?

Thanks,
Doug

------------------------------------------------------------------------------------------
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  LSN 463              email: [hidden email]<mailto:[hidden email]>
voice:  520-626-2824       fax:  [phone]

http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www
Home of: "Microscopy and Imaging Resources on the WWW"

UA Microscopy Alliance -  http://microscopy.arizona.edu<http://microscopy.arizona.edu/>

[Steffen Dietzel]

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Hi Doug,

I have heard the Brilliant Violett dyes are good. Haven't tried them
myself for the lack of time, but I would be interested in results.

Steffen


Am 21.08.2018 um 01:06 schrieb Cromey, Douglas W - (dcromey):

> *****
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> *****
>
> Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.
>
> I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.
>
> The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?
>
> Thanks,
> Doug
>
> ------------------------------------------------------------------------------------------
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  LSN 463              email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www
> Home of: "Microscopy and Imaging Resources on the WWW"
>
> UA Microscopy Alliance -  http://microscopy.arizona.edu<http://microscopy.arizona.edu/>
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Dmitry Ushakov-3]

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eFluor450 works really well in confocal with 405 nm line.

[Alison J. North]

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Hi Doug,

I may have missed this - lots of replies coming through and I'm busy at a course - but I've had the most success with DyLight 405, as long as you use it with either Prolong Diamond (not Gold in this case) or homemade N-propyl gallate antifade.  Alexa 405 is also stable in either Prolong Gold or Prolong Diamond but it's much weaker than DyLight 405 in my hands. I've tested a lot of different blue dyes and the choice of antifade seems to be particularly important with them.

Just my own findings, which I keep meaning to publish somehow but have never had time...

All the best,
Alison



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"Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:

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Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.

I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.

The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?

Thanks,
Doug

------------------------------------------------------------------------------------------
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  LSN 463              email: [hidden email]<mailto:[hidden email]>
voice:  520-626-2824       fax:  [phone]

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