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Dear Jelle,
eosin is fluorescent, I agree, but hematoxylin is not. So a
hematoxylin-only staining might work for you. It does quench
fluorescence of course, as other brightfield dyes, as others already
pointed out.
I would think that you need a lot more molecules of a brightfield dye to
make it visible, compared to a fluorescent dye. Thus quenching is
probably unavoidable.
Concerning the number of fluorescent channels available, 1-Dapi,
2-green-GFP/488, 3-orange-Cy3, 4-near red, Cy3.5 or AF594, 5-far red -
Cy5 or AS 635P, 6-far far red, AF 680 or CF680R, and that is without
spectral or lifetime unmixing. That could be Dapi plus 5 different
antibody stainings. Some want to have a lot of fluorochromes at first
but if the question comes up how many structures they actually are able
to stain in the same sample, the numbers often crumble, because only a
limited number of hosts for primary antibodies is available.
Best
Steffen
Am 22.03.2022 um 13:35 schrieb Jelle Postma:
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>
> Hi all,
>
> Risking a slight departure from the confocal microscopy topic;
>
> Do you know of any compounds that I may use as a non-fluorescent dye
> to increase the contrast in brightfield, in this case specifically of
> PFA-fixated mouse brain slices?
>
> The idea is to make them more clearly visible on a really quite basic
> brightfield system, while at the same time avoiding the introduction
> of any fluorescence, in order to keep the full spectrum available for
> any further desired fluorescent markers.
>
> For example hematoxylin / eosin, or cresyl violet are common
> brightfield stainings but also do introduce fluorescence.
> My searches so far normally end up at sources that review fluorescent
> compounds instead...
>
> Many thanks in advance!
>
> Best regards,
>
> Jelle
>
> ——————
>
> Dr. Jelle Postma
> Light microscopy and image analysis
> General Instrumentation, FNWI
> Radboud University Nijmegen
> Huygens Building, Room HG.01.222
> Phone: 0031 24 36 52199
--
------------------------------------------------------------
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
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