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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) <[log in to unmask]> 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
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