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
