Yishai's injection thoughts:
high pressure -- high rates on the nanoject -- higher volume --
6mm pattern spread
convection device for virus delivery
headplate first, virus injection, 1-2 weeks later; this is so you can remove the glass coverslip nicely
agar and coverslip quickcast
Our ChR2 procedures
We do open dura. We try to make an opening as small as possible directly over the intended injection site. Different surgeons have different preferences as to the tool for the job, but it was usually either a dura pick, or more commonly a 31-gauge needle on a cotton swab applicator. We bend the tip of the needle and it gives a slightly finer tool than the dura pick in the small animals.
The pipettes are pulled on the gravity-assisted puller with settings to give us a long tip which we then break. We check to see if the diameter of the tip falls in a certain range under microscope fitted with a reticule (I believe Arani has reported that range in the document). If they fall outside the optimal range, we discard them and start over. For the good pipettes, we also bevel the tips to around 22 degrees on a Narashige beveler for 2-3 mins. and then check the smoothness of the bevel and to ensure there's no debris in the tip under a microscope in the transgenic facility.
When I inject mice with GCamp (same all speeds and flex/normal), I drill the craniotomy most of the way through the skull, wait 2 min at the brain surface, go into the brain 0.2-0.3mm and wait 2 more minutes, inject 100nL virus over about 4 minutes (0.2nL every 5 secs), wait two minutes after injection, then slowly pull out the pipette. If I remove the pipette to fast, I get virus backwash. I have tried lots of different titers, so I don't have one dilution I use only. I like 1:5 (0.5ul virus and 2.0ul fast green tinted saline).
Nope, that's it. I drill until its optically transparent when flooded with saline and the pipette will go through without breaking. This is the method that Anna prefers, but I'm having issues with it, namely that I can't see the surface of the brain to determine how deep to take the pipette. I think this is why all of my injections were too deep last litter. I'm also concerned about what happens to the piece of bone that the pipette either has to break off or shove into the brain upon entry.
On the upside, the dura remains intact and I can drill a really nice imaging craniotomy (which isn't always the case with complete craniotomies for injections).