[Histonet] eGFP in fresh frozen section combined with CD marker stain

[Gayle Callis]

We are returning to combining GFP and immunofluorescence staining after a
few years of trying it unsuccessfully. 

Question: 

How is the newer eGFP holding up to solvent fixation, acetone or
acetone/alcohol fixation so one can detect another cell (murine CD marker)
population in contrast to the eGFP?  Fresh, snap frozen murine sections
will be fixed with either acetone or acetone/alcohol for the CD markers.  

Previous experience with this fixation needed for CD markers resulted in
GFP losing fluorescence after using these organic solvent. Paraformaldehyde
(PFA) fixation preserved GFP (we are using an improved eGFP now) but CD
markers were a total failure, they hated the PFA. 

Last resort is to use antiGFP, but that defeats the purpose of having it
there, see it GFP glow without having to use immunostaining to detect it.   

 




Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)