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!
You'll probably induce some reactive astrogliosis & microglial activation just by the mechanical trauma. Maybe your mice are also developing an anti-vector immune response. You could try steroid immune suppression for a month after injection. However my preferred method would be to inject the mice as neonates - ideally no later than two days after birth. We see no immune reaction with injections at this age. You'll get more vector spread which may be a problem for - although as a solution, lentivirus vectors tend to spread less. You could lock down expression using a more specific promoter. BW, Simon
You'll probably induce some reactive astrogliosis & microglial activation just by the mechanical trauma. Maybe your mice are also developing an anti-vector immune response. You could try steroid immune suppression for a month after injection. However my preferred method would be to inject the mice as neonates - ideally no later than two days after birth. We see no immune reaction with injections at this age. You'll get more vector spread which may be a problem for - although as a solution, lentivirus vectors tend to spread less. You could lock down expression using a more specific promoter. BW, Simon
I do see the same using AAV, Lentivirus, knife transection or retro-tracer injection. It always results in high GFAP staining and I assume its due astrogliosis after the mechanical lesion.
You could wait until the reactivity decreases if your vector expression and protocol allows that. I did try a M.O.M. (mouse on mouse) kit from vector but poor results. For me, the only solution is to use a non mouse primary antibody.
A course of corticosteroid given over time may clear the reaction.
Have you considered pre-surgery oral or intraperitoneal injection of corticoseroids, although like David waiting it out may be another option.
Hi Shanu,
I have had the same problem with anti-mouse secondaries recognizing microglia. Are you using monoclonal mouse antibodies? If so, you could try IgG subclass-specific secondary antibodies (e.g. IgG1, IgG2a, IgG2b). This has made a big difference in some of my immunostaining experiments.
Peter
Thank you all for the responses. I need to use adult mice because of the specifics of my experimental design. Regarding the corticosteroids, does anyone have any references I could look at? Also, thanks Peter. I'm using an IgG1 monoclonal so I think I'll try the subclass-specific secondary if it has improved matters for you.
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