Hello. I implanted single cannulas bilaterally into the brains of mice to infuse drugs. After behavior tests, what would be the simplese way to validate if the cannulas are in the target areas? I'm thinking about doing cryosection. For cryosection, is transcardial perfusion a must when I don't need to store the brain sections for staining or can I just put the brains in 4% PFA for a day and transfer them into sucrose solution? Any advice would be appreciated. Thank you.
Hi Kwanghoon,
I generally transcardially perfuse the mice first with 0.9% saline and then 4% PFA while the cannula is still implanted into the brain. On this way, the tissue around the cannula will be fixed on that position and it will be easier to visualize the cannula track. At the end of perfusion, you remove the cannula and the brain and postfix the brain in 4% PFA for 24 hours followed by cryoprotection in 30% sucrose solution. After cryosectioning the brain, I float the sections in PBS 1x and counterstain them with DAPI! Let me know if you have more questions and good luck!!
Just to add a proposal for reading the following RG-Thread:
Long-lasting_non-toxic_dye_injection_in_the_brain ?
asked by Gregorio Galiñanes 26.26 University of Geneva, Jan 14, 2013.
cf: https://www.researchgate.net/post/Long-lasting_non-toxic_dye_injection_in_the_brain?
Best wishes and good luck, Regards, WHM
Hi Kwanghoon,
For simplest way to locate your cannula's tip, you even do not need to perfuse the animal and do not need to fix the brain in PFA. You can just extract a brain, freeze it in -20 and start cutting. You need to set a right angle of cutting blade to see the cannula track from top to bottom on the cut surface. And you need a photo camera with good resolution to make pictures of cut surface from short distance.
You can also inject small amount of ink via your cannula after making deep anesthesia. Last helps you to see track better.
We have several published data when we used this way of cannula location:
Article Nitric oxide production in the perifornical-lateral hypothal...
Article Mechanisms mediating effects of nitric oxide on perifornical...
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