11 November 2016 7 9K Report

I am injecting an AAV into the hippocampus of adult mice that upon infection, lead to expression of a protein only in neurons. While I am successful in getting this protein expressed in just my target cells, I am encountering issues during immunos. When I use primary antibodies made in mice, the anti-mouse IgG secondaries are picking up what look like microglia with highly ramified branches popping up everywhere, even far from the injection site. I've checked without using primaries, and the secondaries will pick up the same signal. Blocking with Fab fragments resolves the issue slightly, which has me convinced that I am seeing IgG immuno-reactive microglia. My question is how to prevent this from being an issue to start off.

I see similar artifacts with injections of  AAVs of serotype 5 and serotype 9. I have tried different injection volumes (100-500 nL) and injection rates (10-100nL/min). I have also tried different ages for injection (4-12 weeks) and different expression times (3 or 4 weeks). I am using a 33 gauge cannula to administer the AAV and the the cranial window I am creating is tiny. I am injecting into the dorsal hippocampus.

Out of the 15 or so mice that have been injected thus far, only 2 have been artifact-free, but these same conditions when repeated sometimes have artifacts. Anyone had the same issues or have any suggestions on how to resolve this?

Thanks!

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