[Histonet] BrdU and EM?

[Mezey Szilvia]

 Yes, I have tried with all kinds of concentrations of HCl and 
different times of incubation as well. The BrdU labelling works even 
with 10 minutes of HCl incubation (this is not very consistent, 
though) but the ultrastructure is 'dead' by that time.

 Has anyone got any experience with breaking DNA strands using 
UV light? What's the wavelength that could be used and how long 
does the tissue need to be exposed? My first try was our 
fluorescent microscope (at 360-370 nm), exposure time 1 hour, but 
it doesn't seem to do the job .


 Szilvi Mezey



Subject:        	RE: [Histonet] BrdU and EM?
Date sent:      	Thu, 18 Sep 2003 09:40:23 +0100
From:           	"Edwards, R.E." <ree3 <@t> leicester.ac.uk>
To:             	"Mezey Szilvia" <mezey <@t> ana.sote.hu>


>          Have  you  tried  cutting  down  the  HCl time  to  a  
minimum?????
>                                                                            Richard  
Edwards
>                                                                                             
MRC TOX UNIT....U.K.......
> 
> 
> 
> -----Original Message-----
> From: Mezey Szilvia [mailto:mezey <@t> ana.sote.hu]
> Sent: 17 September 2003 12:54
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] BrdU and EM?
> 
> 
>  Hello All,
> 
>  I'm trying to do BrdU staining (in paraformaldehyde fixed, free-
> floating sections) and preserve the ultrastructure of the tissue for 
> EM at the same time. HCl works fine for denaturing DNA for BrdU-
> ICC but doesn't leave much of the tissue for EM. DNase would be 
> more EM-friendly but doesn't penetrate the tissue enough.
>  
>  Does anybody know a method that could provide a fair enough 
> compromise between BrdU and EM? Maybe by increasing the 
> penetration of DNase?
> 
>  Best regards to you all,
> 
>  Szilvi
> 
> Szilvia Mezey
> PhD student
> Semmelweis University
> Dept. of Anatomy, Histology and Embryology
> Tuzolto u. 58. Budapest, 1094, Hungary
> T.: +36-12156920/3687
> F.: +36-12155158
> E-mail: mezey <@t> ana.sote.hu
> 
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Szilvia Mezey
PhD student
Semmelweis University
Dept. of Anatomy, Histology and Embryology
Tuzolto u. 58. Budapest, 1094, Hungary
T.: +36-12156920/3687
F.: +36-12155158
E-mail: mezey <@t> ana.sote.hu