Do you have access to a 12-15 mL syringe? You can do the transcardial perfusion by hand without the pump (and you would at least want to clear blood from the system before fixing). Simply fill it with cold PBS attach an 18gauge needle, and insert into the heart (under anesthesia). Following the PBS clearing of blood, you could then run PFA through the vasculature to ensure fixation of a specific brain structure without comprising cells. Immersing the brain in PFA will fix the tissue though the penetration will take some time (>24hrs). You risk your structure's cells dying before fixative even reaches them snd thus your analysis will not be under ideal conditions. Whatever you choose, getting the blood out of the system should be a priority, especially for IF as blood will likely cause background signal when you go to image your samples.

yes

Hi Esteban,

I fully agree with Elliot's sound advice. Perfusing is almost always worth it, and mice can be easily taken care of with syringes instead of pumps.

I'll add that we oftentimes need to use different hemispheres from the same brain for lysates and slices. In those cases, we haven't found too many problems by just immersion fixing the half that's destined for slicing. Especially with such a superficial nucleus I think you should be fine with immersion fixation.

BUT there's no question that you'll have higher background and more potential for off target staining if you don't perfuse at all. And if you're already perfusing with PBS, I don't see why you wouldn't just flush PFA after and get the fixation started before you even dissect out your brains....

But how would you control pressure? Don't you need to be careful not to blow up blood vessels?