I am not expert in this field

The clogging of syringes in my experience came from poor cleaning after use, so i recommend cleaning the syringe with the designed cleaning wire. One good system to evaluate if the syringe is clean is that when you fill it with water and expulse the water quickly, it should make a straight continuation of the syringe and not have any angle from it.

Another trick that might help is to apply some positive injection pressure while you go down in the brain, this will result in small amount of viral infection along the needle tract (which if you virus express a reporter gene might help locating the tract).

Thanks, Jonas.  I suppose it'd be helpful if I might briefly describe my current protocol.

 Before each infusion, the syringe needle is cleared & rinsed - using the cleaning wire as you described.  Before loading the syringe, we ensure that fluid will flow coherently from the tip in a straight direction when expelled.  

Then, immediately before loading with viral fluid, the ability for the syringe to fill effectively (without including bubbles in the chamber) is observed using our microscope.  If all is well, we then load with viral fluid - again, under a microscope to monitor for bubbles.  Immediately following loading, we ensure that a small drop of viral fluid (~2uL) will form at the tip of the syringe.  This is also done the moment before infusion into the brain.

Once the syringe is lowered to the intended target, we permit ~5 min of settling time before beginning infusion.  Depending on the target and mouse we're working with, the infusion can proceed rather slowly - after which, we observe another ~5 mins of waiting time before beginning the slow process of withdrawing the syringe from the brain.  

Rinse & repeat.

Despite fairly meticulous care to avoid clogging, we still encounter some.  I anticipate that there's an unavoidable rate at which this will occur, but I wonder if anyone has a protocol that reliably minimizes (or eliminates...) this issue?

Hi Ian,

a couple of questions to clarify the method:

1. are you cutting the dura before going down?

This might be the cause of clogging, i used a tip of injection needle tapped against stainless steel (usually the Kopf table or the surgery plate) to rip the dura and provide direct access to brain tissue. This in my experience was a key procedure to reduce clogging and ensure good targeting of my brain area of interest

2. how fast are you going down?

Again, i have no evidence this impact, but you might try to go faster or slower depending your current speed and see if this is helping anyhow.

Also note that depending where your brain target is it can be that the serynge was not clogged but simply you injected in the ventricule, it happened to me with the anterior thalamic nucleus. I found it out by looking at fluorescent dye injection and noticing a staining on the surround of the ventricule.

Hello, I also have the same problem. I infused into paraventricular thalamus. Do I need to going to the target site quickly or slowly? Another question is often leakage from injection track. It causes contaminated the ventricle and around area. Does anyone have good suggestion to avoid these problems

In our experience, small needles will have a chance of cloggin up. I found two ways to reduce the incidence rate: 1) Was there bleeding during the needle insertion through dura? If yes, then blood clotting will likely cause problems. Removing dura with a sharp needle ahead of the time, draining the remaining blood before proceeding with an injection will help. 2) Bleeding at the site of injection - start injecting immediately after lowering the needle (you wrote:" and I infuse very slowly after waiting periods. "). We did an MRI of mice right after an IC injection and many of them had bleeding right at the site of infusion, which would clog the needle if left unattended for a couple of minutes.

You could also use a larger needle. People use anywhere from g27 to g35 depending on the goals and applications.

Hi everyone - surreal how long ago I posted this question.

To respond to Jonas Hauser's topics:

1. Yes, I ensure the dura are pierced before lowering the syringe.

2. Speed of lowering: depending on whether I'm using a Cre-dependent or independent virus, I adjust it. I've found that - when using Cre-independent viruses, and targeting a rather small & dense nucleus (as I do) - moving as quickly as possible with a small-gauge syringe tip minimizes off-target expression. With Cre-dependent viruses when targeting an anatomical region with highly specific Cre distribution, I move a bit more slowly.

Beyond that, I believe I've gotten to the point where I've optimized the procedure as much as physics allows. For context, I use blunt-tip, 32GA syringe tips attached to a Hamilton 10µL syringe (after testing several). Here's what I do after a fair bit of trial & error:

1. Clear the syringe tip prior to every single injection using one of the wires that comes with each syringe tip, and ensure a test fluid will flow unimpeded before loading with viral fluid.

2. Prior to lowering the syringe: ensure a droplet of viral fluid forms when very small volumes of fluid are infused to make sure the virus will flow.

3. When targeting ventral structures with Cre-independent viruses, get there as quickly as possible.

4. Once at the DV coordinate, overshoot by ~0.03mm, then lift back to target

5. Wait a maximum of 5 minutes prior to infusion; fewer if the target structure is large

6. After infusion, wait 5 minutes for small, dense, ventral structures

7. Raise syringe painfully slowly.

I've also found that viral fluids derived from different core distributors seem to vary in how likely they are to clog even before the syringe ever touches tissue. Now, using exclusively viruses that we make the in the lab, this has been less of a problem.