[Histonet] RE: problem in dual staining of CD4 and FOXP3
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Fri Nov 9 02:08:07 CST 2007
Hello Ann,
Looking at your double staining protocol I noticed that the normal rat
serum blocking is not blocking but making the cross-reaction problem
even worse. Normal rat serum will bind at randomly all over the tissue
section and your last step 6 (donkey anti-rat/AF488) will bind to it.
Therefore I am sure that your whole tissue section is glowing bright
green!
Our research group have recently published a paper in showing
CD4/FOXP3 double staining in human tissues, but we used enzymatic
labels here (De Boer et al., JHC 2007, 55:891-898). The use of
enzymatic labels allows a relatively easy sequential double staining
procedure.
However, immuno fluorescence double staining with two unlabeled
primaries of the same species is not easy. Most promising is a kind of
sequential approach starting with a very high dilution of the first
primary detected by a super sensitive fluorescent tyramide
amplification method. The second primary antibody is simply detected
by a 2-step procedure. No blocking step in between! The rationale
behind this, is the very high dilution of the first primary that is
not detected by the second detection system. See Brouns et al., JHC
2002, 50:575-582. You have to test a dilution series of the first
primary followed by the tyramide-fluo1 detection, then omitting the
second primary and applying the final anti-rat/fluo2 step. From the
dilution series you have find a dilution of your first primary that is
showing a good signal but that is not picked up by the second
detection system.
Good luck!
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
Date: Thu, 8 Nov 2007 08:38:39 -0500 (EST)
From: "FU,DONGTAO" <fudo <@t> ufl.edu>
Subject: [Histonet] problem in dual staining of CD4 and FOXP3
To: histonet <@t> lists.utsouthwestern.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 ye! t. Does a
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