I think the problem is that you are blocking with rat serum.
You wouldn't block with rat serum before your first primary, and you shouldn't for your second primary since both are rat antibodies.
There are several ways you could try to accomplish dual staining of 2 rat antibodies. I think the easiest, since you are doing fluorescent on frozens, is use BD's rat x mouse CD4 FITC (green cytoplasm). It works very well. Ebioscience has AF conjugated primaries, so you should be able to get rat x FOXP3 AF555 or AF594 (red nucleus). Then you could just cocktail them and do single step staining.
Or you could try Probes (Invitrogen's) Zenon labeling kits, and directly conjugate your rat antibodies yourself. (I don't know if they have kits for rat primaries though)
Thirdly, you could try following your protocol for the first primary, adding Biocare Medicals' Denaturing solution to seal the antigen site (since the first is cytoplasmic) then the second would have access to the nuclear site.
1. 5% 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. Biocare Medicals' Denaturing solution (follow manuf instructions)
5. Serum block: 5% normal DONKEY serum 30 min
6. 1* antibody FOXP3 1:100 in diluent 1h RT
7. Secondary AF488 Donkey anti-rat in TBS 1h RT
Melissa A. González Edick
R&D, Cell Genesys Inc.
500 Forbes Blvd
South San Francisco, CA 94080
f (650) 266-3080
Date: Thu, 8 Nov 2007 12:59:10 -0500 (EST)
Subject: RE: [Histonet] problem in dual staining of CD4 and FOXP3
Content-Type: text/plain; format=flowed; charset=us-ascii
Yes, I am looking at T-regulatory cells of mouse. For CD4, I
used AF594(red color), for FOXP3 I used AF488(green color). The
problem for me is for the second primary antibody(or maybe I
should say for the secondary color) of dual staining, it never
worked. I do not know which serum I should use to block the
non-specific staining of the secondary antibody. If you think my
protocol is imcomplete, could give me some suggestions? Many
On Thu Nov 08 12:45:24 EST 2007, "Tarango, Mark"
> Looking at T-regulatory cells, huh? Are you trying to stain both
> in the
> same color? I know Foxp3 is nuclear and CD4 is on the cell
> but two colors might be better. Your protocol looks incomplete.
> Mark Adam Tarango HT(ASCP)
> Histology & IHC Supervisor
> Nevada Cancer Institute
> One Breakthrough Way
> Las Vegas, NV 89135
> Direct Line (702) 822-5112
> Treo (702) 759-9229
> Fax (702) 939-7663
> -----Original Message-----
> From: firstname.lastname@example.org
> [mailto:email@example.com] On Behalf Of
> Sent: Thursday, November 08, 2007 9:13 AM
> To: Histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] problem in dual staining of CD4 and FOXP3
> 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"
>> 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
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