I am working on some retrograde tracing experiments in which I am injecting fluorescent microbeads into a particular brain region and performing electrophysiological recordings in the cells that they trace back to. I'm using the azide free Fluospheres from Invitrogen, and I've had some issues with the beads traveling up the injection tract instead of staying localized in the region I want to inject. I've tried a lot of different things during the surgery (slowing injection rate, preloading saline or mineral oil, slowing needle raise), and these help but don't completely fix the issue. Has anyone tried the Retrobeads from LumaFluor and seen this issue? I'm thinking of switching, but don't really know if the Retrobeads are any better. I would also be interested to know if anyone has compared the health of the cells during electrical recordings in Fluosphere vs Retrobead labeled cells, as that seems to be what LumaFluor is trying to sell on their website.
Hi Marissa. I have not personally done retrobead injections, but have been shadowing a college who has been doing them. What she does is once you've reached your DV is to go about 0.5 further down, and then come back up to your DV before doing the injection (making a sort of "pocket" for the injection). She also leaves the pipette for 5 minutes before retracting it slowly.
I have been trying the ephys with retrobeads and have been having a lot of trouble making seals on those cells. I find that they are extremely delicate and the membranes break very easily while trying to make a seal. I have, however had no trouble making seals with and recording from other surrounding cells that do not express the retrobeads. This paper does ephys recordings with retrobeads Article A Unique Population of Ventral Tegmental Area Neurons Inhibi...
I hope this is somewhat helpful. If you see any tips about the ephys side please let me know.
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