Hello everyone,
I am injecting lentivirus into the hypothalamic region of the mouse brain. I am going in DV -6.05 mm and, more often than not, I have brain matter clearly clogged at the tip of the pipette (~70um inner diameter), which obviously means virus was not injected (500 nl, also visually checked via level of mineral oil-viral solution interface higher up in the glass pipette). I am using a Hamilton 7105 attached to the glass pipette via tygon tubing filled with mineral oil. I have tried slowly putting the pipette in, but results are same. I also retract 0.2 to 0.3 mm ventrally after hitting the target DV.
How can I avoid the clogging?
Any help/tips would be greatly appreciated.
Thank you!!
PS: not sure if relevant, but I'm finding that I go through the ventricle, because there is alot of backflow once pipette is extracted, much more than the 500 nl I programmed for injection.
Slightly unrelated... but 500 nL? That's a pretty massive amount. A sphere with a diameter of 1 mm has a volume of 65 nL... 500 nL is a sphere with a diameter of 2 mm. That's a pretty big hole to be pushing into the brain.
A) I think you want to make your pipette tip a bit bigger. 100 um at least.
B) If your injection system is put together properly, even a severely blocked tip should get cleared when the pressure comes on.
I agree with William that the volume is quite large... I inject in the hypothalamus at ~ 3.0 DV. I have issues with blood clogging the pipette occasionally, but I inject at 41.4nL intervals, and by the 3rd injection, any block is cleared away because of the pressure I have built up in the pipette.
Do you wait 10 mins before retracting your pipette to allow for the virus to diffuse? And do you retract slowly?
I agree that your volume might be too large. You could try injecting some dye following your method to better understand if the volume is appropriate and if it diffusés where you want. I found it more successful to do 2-4 injections in the area rather than at one spot to avoid trauma. The final key step is to retract the pipette slowly and make significant pauses when you target the hypothalamus. Usually we do 3 stops of 2 mn each on the way up. Hope this helps
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