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