Hi guys,
How do you mount the dissected hippocampus onto specimen disc for horizontal sectioning?
Currently, I am trying to set up a rig to recording field EPSPs using mouse horizontal slices. However, I found it extremely difficult for me to stick these hippocampus tissues onto specimen disc covered with thin layer of crazy glue. I followed some videos online, cutting off both ends of individual hippocampus and facing one with the other. The mouse hippocampus is so small and soft that I failed most of the time in last two weeks or so.
I am using 7-8 months old mouse and sucrose cutting solution.
Any suggestion will be high appreciated.
Hi Fading,
I hope this helps, when I have to do acute slices of delicate parts of the brain (like the olfactory bulb for instance) I prepare some slicing solution with 1% agarose on it to make a gel (in a petri dish, for convenient storage in the fridge). Then I cut blocks of this gel of appropriate size with a scalpel (out of the petri dish) and stick them with superglue onto the specimen disc of the slicer in appropriate positions, so that when I later stick the dissected brain, the delicate parts have support against the agarose blocks. This dramatically reduces sheer forces during slicing of delicate brain structures.
So heres what you could try: cut a wedge shaped block of agarose and stick it to the specimen disc ramping up (not too steep) and cut a groove in the middle of the ramp. Then stick just one end of the hippocampus to the bottom of the disc so that the rest rests on top of the groove in your agarose wedge (ramping up). Then try if the slicer can slice the part of the hippocampus surrounded by agarose without problems. Thew will not be perfect horizontal slices but might solve your problem.
Good luck!
The agarose idea is good, and we've used it successfully as well. It is more time consuming and most of my students currently use the approach below because of that.
With older animals it's often better to perfuse them transcardial with cold ACSF before cutting, and you can also immerse the brain in cold ACSF prior to mounting on the stage. We make a small 30 degree angle cut on the dorsal surface to give a flat gluing surface, and use Vetbond glue. We always blow on the glue to help dry it in a second or two, and then immerse the brain in cold ACSF (4 degrees). If you cut at this temp the brain should be fine.
Thank you, Juan and Brain for the idea of using agarose as supporting material and other useful tips. A guy in our institution suggested to do whole brain slicing first and dissect out hippocampus afterwards.
I will try all these slicing methods to see which works bests for LTP induction.
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