visualising EGFP in vivo

Branwen Morgan b.morgan at garvan.unsw.edu.au
Tue Jun 15 20:00:36 EST 1999

Hi all,

I was wondering if anyone else has had problems visualising GFP/EGFP in tissue?
I am using the pIRES-EGFP vector for microinjections to make my transgenic
lines.  I have recently sacrificed a positive F1 mouse (genotyped by PCR to
EGFP) to look at brain tissue for expression. Although it appears I have
fluorescence in some neurons, a similar pattern can be seen in the control
mouse. After reading other emails mentioning that ethanol negatively
affects the GFP, I am doing cryostat sectioning on fresh tissue.
Slides are then washed in 1x PBS 10 mins and coverslipped (glycerol/PPD) or

There was about 24 hours between tissue collection and microscopy for the
fresh positive tissue.  However, the control brain had been at -70deg for 3
weeks (frozen on dry ice).

Previous mouse brains had been paraffin embedded, dewaxed etc...and I saw
nothing. We attributed this to the alcohol dewaxing steps, and when I did
IH using antibodies there was some expression - so I know my construct is
expressing and that my PCR genotyping is not giving false positives.

I will try that sodium borohydride wash next to hopefully reduce
autofluorescence ....but does anyone else have any other suggestions?

Thanks very much,

Branwen Morgan

PhD Scholar
Neurobiology Programme
Garvan Institute of Medical Research
384 Victoria St                           Ph   : 9295 8305
Darlinghurst                              fax  : 9295 8281
NSW 2010
email:   b.morgan at garvan.unsw.edu.au

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