Is it possible to observe fluorescent signal in fresh cryoslices prepared from mice with stereotactically infused viral GFP?
I would just want to have a preliminary verification that there is a GFP signal present without the need to do any post-fixing or staining.
Hi Samuel Kohtala ,
Please be aware that the native GFP signal from snap-frozen brain sections will probably not be as good as from perfused (fixed) brains. The GFP might leak out of cell bodies and projections. You will still see fluorescence around the site of injection, but maybe not individual cells. It's probably OK if you just want to check the site of injection though. I think it also depends on whether your GFP is free or fused to another protein...
There is a paper describing a method to reduce this leaking effect: basically, after cutting the slices, you post-fix them in 4%PFA warmed at 37°C, without letting the slices dry out. It can be a good compromise if your signal is not good enough for what you want: https://www.nature.com/articles/s41598-020-71493-x
Depends on the Signal to Noise (S/N) from the GFP, its distribution and concentration, depth at which the signal originates (scattering of the emission light), the penetration depth of the excitation light, etc. If you have access to a multi-photon system with a suitable sample handling setup, it should work without much issues.