[Histonet] RE: problem in dual staining of CD4 and FOXP3

[C.M. van der Loos]

   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