[Histonet] Effect of Freezing on Unfixed GFP

[Andrew Coleman]

We have performed lentiviral injections with a turboGreenFluorescentProtein
(tGFP) into the brains of rats for an RNAi experiment. We did not fix the
tissue but froze in isopentane on dry ice and stored the tissue at -80 deg
Celsius.

We want to use some sections for a reference of the injection site (GFP
fluorescence) relative to the brain morphology (Neurotracer staining) in
order to selecitvely laser microdissect (LMD) infected cells from other
sections.

Our initial studies showed that fixation of sections on slides in 4%
paraformaledhyde (PFA) at 4 deg C for 5 minutes provided tGFP flourescence
comparable to that found in perfused tissue, but lately we see very weak or
no fluorescence with this procedure, even when increasing the time.

The only difference I can think of that we saw best results soon after the
brains were removed (ie 1 to 2 weeks) and now we are having trouble
visualizing the tGFP ~2 months of the brains being stored at -80 degrees
celsius.

Does anyone have experience with or an explanation of fluorescence
decreasing with a frozen, unfixed GFP over time?

In the future we plan on doing these experiments including a perfusion with
a low conc of PFA (to preserve RNA integrity) but for now we are trying to
figure out how to identify the GFP-positive cells in the animals we have
already performed surgery on. Another option would anti-tGFP IHC, but we
would prefer at this point to use on slide-fixed sections as a references
for our LMD.

Thanks!  -Andrew