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Nile Red has a long spectral tail and can exhibit spectral shifts under
various conditions. Have you considered Prodan or similar as an
alternative?

Craig Brideau
On Sep 8, 2014 8:04 AM, "Shane van Breda" <[log in to unmask]> wrote:

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> 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.
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>
> Dear group,
>
> I would like to try the following and I am hoping someone can give me
> insight.
>
> I will be incubating M. tuberculosis in presence of an antibiotic for 24
> hours. I will
> use it at the determined MIC and 2 x MIC.
>
> I will remove the cells, centrifuge to obtain a pellet, rinse and
> resuspend it. I will
> stain with Draq 7 (3uM) and allow for the reaction to take place at room
> temperature for 30 min.
>
> Once stained with Draq 7, I will centrifuge and concentrate the pellet
> (100ul) and
> spot it onto Poly - L - Lysine slides (20 ul). I will fix and sterilise
> the slide using
> formaldehyde fumes (25 % v/v) over night (previously established protocol).
>
> Next, I would like to rinse the slide, allow it to dry, then stain the
> slide with Nile
> Red, followed by rinsing.
>
> I want to view Nile red at Ex ~ 488nm, Em ~ 525 and Draq 7 at Ex ~ 633 or
> 647 ,
> Em ~ 695 LP.
>
> My concern is that there will be an overlap with Draq 7, but I am not 100
> % sure?
>
> Thanks in advance
>