hi,
1. no idea.
2. yes. if they proliferate.
3. just treat with PBST (Tris-buffered PBS) before your adding of antibody.
4. possibly..just notice some BrdU positive cells could be microglia..be careful of the leakage..and if you dont have a GFP-rat, have you considered of using astrocytes from mice for injection into the rat brain?


2008-09-16 



tf 



发件人： Nejat Yilmaz 
发送时间： 2008-09-16  14:24:11 
收件人： histonet@lists.utsouthwestern.edu 
抄送： 
主题： [Histonet] BrdU incorporating and staining 
 
Dear Netters,
We are setting up a cell therapy model in neonate rats by injecting  primary 
cultured astrocytes intracerebrally.
My questions are:
1. Is it possible to incorporate BrdU in all cells by adding it 10 uM 
concentration into culture media from beginning of the culture?
2. If so, can we demonstrate BrdU in confluenced cell nuclei by using IHC or 
IF on petri culture dishes? (Fixed with %4 PF for 20 min.)
3. Should we use detergents or any other agents for facilitating penetration 
of the antibodies in to the nuclei, any special protocol suggestion?
4. If we accomplish this difficulties, can we demonstrate these labelled 
donor cells in host animal brains a few weeks later by using IHC or IF?
And the last thing I have to point out is: unfortunately we don't have any 
possibility to obtain knock-in animal or vector virus treated cells.
We would be really appreciated your helpful comments.
Necat Yilmaz 
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