[Histonet] problem in dual staining of CD4 and FOXP3
Tarango, Mark
mtarango <@t> nvcancer.org
Thu Nov 8 13:16:35 CST 2007
I didn't look closely enough at your protocol. I think you should omit
step 4 of your protocol. Maybe when you trying blocking with rat serum
the AF594 gets eaten up, since it is an anti-rat antibody. It might
just go into the serum and wash away.
...Wait a sec, you said you were having problems with the second primary
antibody.... I don't know what to say, try direct conjugates.
Sorry I can't be of more help.
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: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
FU,DONGTAO
Sent: Thursday, November 08, 2007 9:59 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] problem in dual staining of CD4 and FOXP3
Hi, Mark,
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
thanks,
Ann
On Thu Nov 08 12:45:24 EST 2007, "Tarango, Mark"
<mtarango <@t> nvcancer.org> wrote:
> 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
> membrane,
> 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: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
> FU,DONGTAO
> Sent: Thursday, November 08, 2007 9:13 AM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] problem in dual staining of CD4 and FOXP3
>
> Hello,
>
> 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
> appreciate.
>
> Ann
>
>
> On Thu Nov 08 08:38:39 EST 2007, "FU,DONGTAO" <fudo <@t> ufl.edu>
> wrote:
>
>> 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
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>
>
>
> 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
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> "EMF <nvcancer.org>" made the following annotations.
>
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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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"EMF <nvcancer.org>" made the following annotations.
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