Hello everyone,
I am in the doing viral injection (hamilton and automatic pump) with AAV-mcherry targeting piramidal neurons in mouse aging from P40 to P50. After 2 to 3 weeks, I performed optogenetics stimulation, then after having the brain removed, soaked in formalin and fixated for at least 48h, I checked the viral expression with fluorescence microscope.
Here's the point: out of 10 trials, 4 times I didn't find any trace about the viral expression. I have checked the whole brain, but still zero. We are talking about zero level expression. On the other hand, in 6 experiements I have strong and proper expression.
In my experimental routine, I had 2 injections per day (2 mice). On the same days, I withdraw the virus out of the same small eppenderf tube each time for one animal. Between the two viral injections, the virus is kept soaked in ice in a dark water proof container.
I am sure the virus was injected (I saw the virus level in the glass capillary lowering). As always, I have waited 10 min before needle removal.
What could have happened in those 4 trials with no expression? Is the virus contained in the eppendorf damaged (at least in some of them)? I really don't know what to think right now.
Any help would be much appreciated.
I can imagine your situation is a bit frustrating! Science can be a bit of a rollercoaster ride sometimes, huh? Let's try to figure this out together.
So, you're noticing that some of your mice aren't showing the viral expression you're expecting. There are several things that might be going on.
Little Variations in the Injection Site: Even with a steady hand, there's a chance that the injection site changes just a teeny bit from one mouse to another. Sometimes, this slight change can make a big difference. Double-check your coordinates and keep your process as consistent as you can.
Virus Might Not Be Happy: Viruses, especially AAVs, can be quite sensitive. They don't like changes in their environment too much. Even though you're keeping them on ice, going through freeze-thaw cycles repeatedly or being exposed to light could be making them less effective. If possible, you might want to consider dividing the virus into smaller portions that can be used up in one go
Mice Being Mice: Each mouse is an individual and may respond differently to the viral injections. Maybe it's something about their biology, or even something like an immune response. It's tricky, but it's part of the process
Equipment Playing Up: Sometimes, your tools might not be cooperating. There might be an occasional blockage in the needle, or the virus might not be making it to the target site properly. These things happen, so it's worth keeping an eye on
Viral Spread and Clear-Out: The virus might spread in the brain or be cleared out by the mouse's immune system at different rates. This could be causing some inconsistencies in your results
It's a Waiting Game: AAVs can take their sweet time to start showing effects. If the timing isn't just right, you might miss out on the expression you're looking for.
Consider running some smaller tests to find out what's going wrong. You could try a different virus and see if you're getting the same problems. If it's an issue with the virus, this should give you similar results. If it's something about how you're doing the injections, then changing up your method or tools might make a difference.
It's very odd that in your failed experiments you are getting *literally zero* expression of the AAV as opposed to weak expression (scattered red cells). Do you have records about which mouse was injected on which day? You say you did 2 mice per day and you have 6 mice with expression and 4 with no expression - do those 4 failures represent 2 days on which zero animals worked? Because if that's the case, I think it makes sense to consider things like the virus having been inactivated/destroyed, or a major problem with the equipment setup.
The other thing that comes to mind is the fixation and imaging/detection. Did you perfuse these mice and then post-fix in formalin, or go straight to drop fixation withour prior perfusion? Also, are you imaging the mCherry that is present in the tissue, or using antibody-based detection to add another fluorophore onto it? Consider possibilities like major difference in fixation quality/time (= more protein denaturation = loss of endogenous fluorescence) or photobleaching (could any of these slides have been left out exposed to light for overnight or longer?) Finally, is there any possibility that there was an issue with the imaging step? (Did you swap from a slide that had mCherry on it to a slide that should have had and didn't, to directly confirm that the microscope settings that had just worked to visualise mCherry saw nothing?) I ask because I have done SO MANY things like leaving a shutter closed or the wrong filter set in place...
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