GFP and BrDU staining

Michael Howell mhowell at MED.UNC.EDU
Fri Jul 12 13:49:47 EST 1996


To anyone interested in BrDU and GFP-
    I have been trying BrDU stianing in cells expressing GFP.  I have 
found that the GFP signal is lost following ethanol or methanol fixation.   This may 
be due to diffusion of the GFP from the cells before being fixed.  However, I have 
also seen a loss of GFP signal following a 10 minute fix in paraformaldahyde 
(4%)followed by a 10 minute incubation in methanol.  This loss of GFP signal appears 
to be due to methanol treatment, as the loss of GFP does not occur when cells are 
fixed in paraformaldahyde and permeabilized with saponin.  In addition, immunostaining 
for GFP with a polyclonal antibody against recombinant GFP and an AMCA secondary 
appears to be stable, even when the GFP signal observed with FITC optics is lost 
following this methanol treatment.  

	The bottom line is that I think a sequential immunostaining for GFP followed 
by immunostaining for BrDU by the conventional 2N HCL denaturation method can work.  I 
have found that at least AMCA is not effected by the methanol, 2N HCL or Na borate 
buffers used during conventional BrDU staining. 

	One last note-has anyone out there tried nuclease treatments instead of the 
acid denaturation to expose the BrDU antigen?  There are many papers describing this. 
 Also, has anyone tried BrDU antibodies that do not require a denatured DNA substrate?
I have seen references to a monoclonal named BU-1 I think???  These approaches may 
also work.

Doug Howe
UNC-Chapel Hill
Neurobiology



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