Loss of GFP Fluorescence
gjones
Posts: 12
Hi All,
With all of the brains we've been clearing, we're noticing that all of the endogenous GFP leaves the cell and is somehow instead staining everything but the cells that originally expressed the GFP. This has happened every time so far and we've tried making new solutions with brand new reagents, as well as just using passive diffusion to clear the tissue instead of the tissue electrophoresis, however each and every time we image the sample, the GFP has left the cell and is instead sitting in the surrounding tissue. We've tried this now on a GAD67 and Thy1 models with the same result. The tissue clearing itself appears to work perfectly, just not the imaging/retention of GFP.
Does anyone have any idea what could be going on??
With all of the brains we've been clearing, we're noticing that all of the endogenous GFP leaves the cell and is somehow instead staining everything but the cells that originally expressed the GFP. This has happened every time so far and we've tried making new solutions with brand new reagents, as well as just using passive diffusion to clear the tissue instead of the tissue electrophoresis, however each and every time we image the sample, the GFP has left the cell and is instead sitting in the surrounding tissue. We've tried this now on a GAD67 and Thy1 models with the same result. The tissue clearing itself appears to work perfectly, just not the imaging/retention of GFP.
Does anyone have any idea what could be going on??
Comments
Do you perfuse the animals? Do you leave the samples in the hydrogel solution for few days before polymerization?
Or maybe you have problems with formaldehyde fixation.
I had no problems using 100ml 16% Paraformaldehyde E15710-S ( http://scienceservices.eu/en/16-paraformaldehyde-formaldehyde-aqueous-solution-em-grade-different-packing-units.html ) and also the specified reagents (Bio-rad acrylamide and Bis and Wako initiator).
For the next couple of samples i prepared my own 16%pfa I will tell you if I have any problems.
Does the hydrogel polymerization occur normally? The polymerized gel should be of jelly consistence.
I suppose it's not your problem because you say that clearing appears to work perfectly, but are the brains transparent enough after clearing? If they are not well cleared you will have scattering and the perception that the fluorescence is unspecific.
Btw, I use Thy1-eYFP line H following the original protocol and I haven't had any problems.