I'm currently doing BDA (biotinylated dextran amine) injections into the motor cortex for anterograde axonal tracing. My BDA is tagged with Alexa 488 and Alexa 564. The biggest issue I'm having at the moment is that my BDA is staining the pia mater of my brain and appears to be going everywhere! I think it's contributing to some of the vessel staining I'm seeing as well.
I've tried varying speeds and amounts of injected BDA. Pulling the needle out slowly, but none of this seems to be really reducing the spillage.
I've attached a picture so you can see the staining all along the edge of the brain.
Has anyone who has done this procedure before encountered this problem?
Are you pressure injecting or iontophoretically injecting BDA? If the former, what is the volume you are injecting? It is possible that there will be tracer leakage out of the penetration and around the skull a little time post-surgery considering you are injecting a cortical, or superficial structure. I have seen similar cases with 'autofluorescence' of pia mater only when I pressure inject FR and a number of AAVs.
I'm doing pressure injections of either 100 nL or 200 nL over the course of 10, 20, or 40 minutes. I leave the needle in place for 10 minutes before withdrawing it. I hadn't considered that it could be autofluorescence.
As Glenn inferred, injecting via iontophoresis might resolve some spillage issues. There would be smaller injection sites, smaller tracts, and potential leakage from the pipette can be partly mitigated by using negative retention currents while raising and lowering the pipette.
Another thing is that BDA (and other dextrans like Fluororuby) seem to be picked up by the vasculature even when injected deep into the brain, or so it has seemed to me. Have you considered tracing with PHA-L? I assume you're going for anterograde tracing.
I hadn't considered PHA-L. My PI recommended BDA so I started there. I'll definitely look into that.
Honestly, the more pressure injections I do the more appealing iontophoresis seems. Thanks both of you for the advice!
I would definitely consider try iontophoresis to inject BDA. It is a clean method that allows you to target subcortical structures without leakage. All you need is a stimulus isolator to set the current level and a pulse stimulator.
I would also suggest injecting BDA with iontophoresis. Using anterograde tract-tracing with PHA-L also is a good alternative. If you move for PHA-L tracing, I would strongly suggest a primary antibody against PHA-L raised in rabbit. Rabbit anti-PHA-L performs better than goat anti-PHA-L.
Hi Katherine, have you noticed any penetration through some arteries during the lowering of pipette? The picture you show is so peculiar in that vessel staining spans almost all the hemisphere but only takes the color of correspondig BDA-Alex tracer. And yes I agree with the iontophoresis injection of BDA which gives you quite a clear background. I visualise BDA with DAB brown staining.
I've switched over to iontophoresis but I'm still seeing vessel staining. Sorry that the picture is a little dark.
I'm doing 5 minute injections with a current of 5 uA, alternating 7 seconds on and 7 seconds off with pulled glass pipettes of 20-50 um. My perfusions are good with no remaining blood in the vein. I reverse the polarity of the current when I insert my needle into the brain.
I'm really unsure of what I'm doing wrong.
- Badges
- Science topic
- Similar topics
- Biological Science
- Neuroscience