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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet