So I have been performing stereotaxic surgery on VGAT-Cre mice. the virus I am injecting is a double floxed hm4d Gi - mCherry reporter. Whenever I finish infusing 250 nL of the virus into the mouse brain, I like to check if the needle is clogged or not by manually pushing the needle forward to see if the water inside the tubing will come out. Note that there is a block of air before the water. So I get surprised when I see a drop or two of liquid before all of the air is pushed out... What do y'all suppose it is? At first I feared it was the virus itself, but that doesn't make sense because my injection needle should have deposited supposedly 250 nanoliters of the virus into the brain already, right?
Hi,
it can be a few things. First, in which area do you perform the injection? Usually you will need to make a delicate withdrawal of the syringe with long pauses before completely removing it from the brain. If you don't you might actually collect some of what you deposited, or some CSF.
Second, you mention the air block, is it located at the end of your 250 nl in the syringe? If so, that would be dead volume. Finally, did you do a control experiment to see if your injection works (i.e., injecting into an eppendorf, with the machine so without pushing manually)? Are you using any methods of verification to ensure that you indeed injected your 250 nl? 1-2 drops could easily represent the whole volume!
Hi! Thanks for your response!! I appreciate it! I performed the injection in the VTA, and I wait five minutes after infusion before slowly removing the injection needle. in addition, I have a machine apparatus that controls the pushing out and pushing in of my syringe. The syringe is 10 microliters in total, and is connected to a long tubing, which is then connected to another injection needle. the air block exists within the tubing so I can clearly visualize if liquid is being pushed in/pushed out. I hope that makes sense. And I did perform a control experiment; and that’s the odd part! When I withdraw 250 nanoliters, and I try infusing, for the first two to three minutes, it appears as if nothing is coming out. But this could also be because the amount that is being infused is so tiny!! So I’m not too sure myself.
I think you should do more stopd on your say up (2 mn after injection then go up a bit wait 2 mn etc).
For the rest it sounds like you have dead volume. The VTA can probably handle a bigger volume did you try diluting your solution? You could also used a colored solution for your test. I hope this helps
you're welcome, and sorry from the typos, typing from my phone clearly was not the best choice!
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