Re: DNA FISH and IHC protocols? **tech support response**

[Roy Edward-2]

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Hi Josh,
Greetings from rainy-summer Britain..

I saw a paper that combined DNA FISH and (immuno-)fluorescence imaging
that might be helpful:

Visconti, R.P., et al. "An in vivo analysis of hematopoietic stem cell
potential: hematopoietic origin of cardiac valve interstitial cells."
Circulation Research 98.5 (2006): 690-696 (from MUSC.edu
<http://musc.edu>).


In essence, they used FISH to confirm by y-chr probe the male mouse
recipient cells from GFP+ female donor stem cells where the latter were
shown to localise to the interstitial valves of the recipient mouse heart
tissue sections and also not be a result of cell fusion.  The reason I
stumbled on this is that they used DRAQ5 for the nuclear counterstaining.
This has been shown to readily withstand the repeated exposures (see
Martin et al. 2005) that would be required from the two steps, it is
spectrally distinct from the Cy3 and GFP emission signatures and does not
risk the UV-activated green-shift that has been seen with DAPI (see Jez et
al. 2013 and Żurek-Biesiada et al. 2013) that might interfere with second
step's imaging channel choices.

However, they did not utilise antibody probes in the IF part of the
protocol but the procedure they use would appear not to preclude that as
it looks fairly standard, apart from any additional blocking step. Choice
of mountant would be important - a removable one (thermo-reversible,
perhaps) may be highly beneficial for the two-step procedure they describe
to permit lifting of the coverslip non-destructively after imaging of GFP
and prior to the FISH probing.

I hope that helps a little.
Kind regards,
Roy

Roy Edward
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E: roy(at)biostatus(dot)com

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Date:    Fri, 8 Jul 2016 20:16:58 +0000
From:    Joshua Zachary Rappoport <[hidden email]>
Subject: DNA FISH and IHC protocols?

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Does anyone know a good source for info about DNA FISH (to count genomic
loci) combined with Immunofluorescence to identify tumor cells from
formalin fixed paraffin embedded tissue?

Thanks so much!

Josh

Joshua Z. Rappoport PhD

Director of the Center for Advanced Microscopy and the Nikon Imaging
Center at Northwestern University
Northwestern University Feinberg School of Medicine
303 E. Chicago Avenue
Chicago, IL 60611
[phone]

http://cam.facilities.northwestern.edu/
http://nic.feinberg.northwestern.edu/

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