I am performing stereotaxic injection of an AAV8 serotype into mice and then immunofluorescent staining for target antigens in free-floating brain tissue. The surgeries appear to go very well and the mice recover normally. However, when I complete the immunofluorescent staining of my target antigens, there is an *extremely* high fluorescent background signal and a complete (or nearly complete) absence of detectable specific signal for my antigen, but only in the vicinity of my injection site (e.g. if I inject into the cortex, the problem is localized to the cortex but not deeper areas; if I inject into the NAc, the problem is specific to the NAc but not the cortex)

Procedure: I perfuse ~4 weeks post-surgery via transcardiac perfusion with saline (0.9% NaCl in ddH2O) and 4% PFA, followed by an overnight post-fixation with 4% PFA. The brains are cryoprotected in 15% then 25% sucrose and frozen on dry ice, then sectioned at -20C on the cryostat. Tissue is permeabilized in PBS-T (0.3% Triton X-100) and blocked with normal serum for the same antibody (5% up to 10%). I have tried incubation in primary antibody overnight at +4C and longer periods (3 days at +4C up to 7 days at +4C). Secondary antibody has been tested at several concentrations and for either 1, 2, or 3 hour incubation periods (RT, shaking).

My initial suspicion was some kind of contamination/infection resulting from the surgery/capillary, however, I have been extremely mindful of keeping everything as sterile as possible and the problem persists across numerous surgeries and litters. I am now wondering if the problem could result from an immune reaction to the virus itself, or another issue. This problem doesn't seem to occur in all mice, but does in a significant portion of them.

If anyone has encountered similar problems or has suggestions for troubleshooting, any help would be greatly appreciated :)