[Histonet] RE: Over lapped flouresence in double staining

From:"C.M. van der Loos"

   Dear Dr. Sohail,

   You  didn't  write  us  what detection system was used for this double
   staining.  The  fact  you observed overlap of SMA and vWF (which isn't
   very  likely  indeed) makes your detection system a bit suspect. To my
   opinion  the most straightforward (and safe) ways to doublestain these
   antibodies are:

   A:  cocktail  of  SMA  (mouse) + vWF (rabbit), followed by a secondary
   cocktail composed of goat anti-mouse/FLUO-1 + goat anti-rabbit/FLUO-2

   B: cocktail of SMA, clone 1A4 (mouse IgG2a) + vWF , clone F8/86 (mouse
   IgG1),  followed  by a secondary  cocktail composed of goat anti-mouse
   IgG2a/FLUO-1 + goat anti-mouse IgG1/FLUO-2

   Lots of success!

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   Date: Tue, 18 Apr 2006 19:00:05 -0700 (PDT)
   From: sohail ejaz 
   Subject: [Histonet] Over lapped flouresence in double staining
   To: [1]histonet@lists.utsouthwestern.edu

   I  am doing double staining of endothelial cells and smooth muscles of
   the blood vessels with vWF and SMA.

      While doing double staining I have encountered overlapping of  both
   antibodies  and  I  cant  see  a  clear  differentiation  between  the
   fluorescence of these two antibodies.

     Could you please guide me how to over come this problem.


     Dr. Sohail


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