[Histonet] problem in dual staining of CD4 and FOXP3

FU,DONGTAO fudo <@t> ufl.edu
Thu Nov 8 11:13:22 CST 2007


  Can anyone give me some suggestions on my case? I know a lot of 
people here have a lot of dual staining experiences. I did dual 
staining on 2 antibodies from different species in the past. They 
worked well. But using 2 antibodies from same species is my first 
time try and I met a big problem. Any suggestions I will be very 


On Thu Nov 08 08:38:39 EST 2007, "FU,DONGTAO" <fudo <@t> ufl.edu> 

> Hi, all
>   Thank you first for giving me some good suggestions on 
> thick-section question I posted last time. Now I met another 
> problem when I did CD4 and FOXP3 dual staining using murine fresh 
> frozen spleen. If I did single staining, both antibodies worked 
> very well. However if I did dual staining, I could only get good 
> result from the first antibody. The second one did not work at 
> all(I mean no specific staining). I used CD4 from BD(rat 
> anti-mouse). FOXP3 I used from ebioscience, also rat anti-mouse. 
> I think there might be something wrong with my serum block(I used 
> normal rat serum block) before I added secondary antibody. Or any 
> other serum block I need to add to decrease the non-specific 
> binding which I have not done yet. Does anyone can give me some 
> suggestions according to my protocol below? How can I get 
> specific staining of the secondary (primary)antibody?  Thank you,
>   Below is the simple protocol I used for dual staining:
>   1. 2% normal goat serum block 20 min
>   2. 1* antibody CD4 1:750 in diluent O/N 4C
>   3. Seconday AF594 GT anti-rat 1:1k in 1xTBS 1h RT
>   4. Serum block:  5% normal rat serum 30 min
>   5. 1* antibody FOXP3 1:100 in diluent 1h RT
>   6. Secondary AF488 Donkey anti-rat in TBS 1h RT
>   Use 1xTBS as wash buffer. Before staining, fix tissue in -20C 
> Aceton for 5 min, then airdry.
> Ann Dongtao Fu MD Ph.D
> Lab Manager
> Molecular Pathology core
> Dept. of Pathology
> University of Florida
> 1600 SW Archer Road
> Gainesville, FL 32610
> Lab Phone: 352-273-7752
> FAX: 352-273-7753
> Rm: D11-50
> _______________________________________________
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> Histonet <@t> lists.utsouthwestern.edu
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Ann Dongtao Fu MD, Ph.D
Lab Manager
Dept. of Pathology
Lab phone: 352-273-7752
Lab FAX: 352-273-7755
Lab address: D11-50
PO Box: 100275
1600 SW Archer Road
University of Flodrida
Gainesville, FL 32610

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