Does the GFP ab label neurons in mice that were not injected with any viral construct? Or in distant brain regions? Were the sections washed enough times between primary and secondary incubations? And then enough before mounting? Are the 2ndary antibodies bad? Was correct blocking solution used? Just thinking of most simple explanations

Just to clarify, if you already have GFP expressed, why do you need the GFP antibody? You should be able to see the GFP as is. In any case, it sounds to me like you might have a lot of background staining (since you see a "GFP" staining in WT mice), maybe you should try different concentration of your antibody. Also, have you tried different secondary antibodies?

Suggested experiments for determining if problem source is: primaries, secondaries, GFP cell marker misdirected to wrong cells or, virus. Primary Ab omissions are always useful to rule out/in some problems.

1) GFP primaries on sections from untreated wild type mice, secondaries as usual (2 slides, one rabbit and one chicken)...do you get label?

2) Primary omission on sections from untreated wild type mice, secondaries as usual (2 slides, one rabbit and one chicken)...if label above is it just due to secondary?

3) GFP primaries on sections from untreated (no virus) GFP mice, secondaries as usual (2 slides, Rb and Chicken).....glia and neurons?

4) Primary omission on sections from untreated (no virus) GFP mice, secondaries as usual (2 slides, Rb and Chicken ).....if neurons label in 3, is it secondary?

5) and 6).....as above 3 and 4 but GFP animals with virus injections (4 more slides total)

Notes: 1)use all fresh buffers and blocking solutions. 2)If slides are dear, just do 1-6 using only one or the other (Rb or chicken) primary Abs with its secondary Ab.