2 replies to this topic

[Delta]

Hi,

I have a transgenic line of mice which express my protein of interest as well as a GFP reporter. I'm taking fixed frozen sections from these mice and trying to localize expression of the vector with fluorescent microscopy. However, I'm seeing what looks like GFP positive cells in the (negative control) wild-type mouse as well. I've repeated it with a couple mice so I'm confident it's not just a matter of grabbing the wrong mouse.

The cells appear to be relatively large and in the midbrain/brain stem areas. Has anyone experienced this before?

Unfortunately my GFP expression in the transgenic mice is seemingly low as well (and in similar areas to where I'm seeing it in the WT) so I'm not sure if I'm simply seeing normal "background" levels of fluorescence or something else. The vector is pIRES from Clontech.

Thanks

[gfischer]

A few thoughts:

For the wild-type mice:  Is the fluorescence present in any other channels?  Neurons do have a tendency to autofluoresce in the green and red channels.

For the transgenic mice:  Fixation can sometimes quench/mask native GFP fluorescence in my experience, although not usually in frozen sections.  Do you have a GFP antibody you can use for IHC to double check the expression?

[Delta]

Hi gfischer,

I haven't tried looking for signal on any the channels in the WT mice. I will give that a try.

I just recently learned that apparently PFA can denature GFP and shift it's emission spectrum -- similar to what you are suggesting. The mice are perfused with PFA and brains post fixed  before freezing so I suppose that could be an explanation?

Regardless, your idea to use an anti-GFP antibody is exactly what I have been trying to avoid (because no, I don't have one and would rather not spend money on it) but it seems like the only course of action at this point.

Thank you for your thoughts on the matter.