Dear research community,
after receiving your helpful answers to my initial post (in quotes below), I would like to offer the method that we have used since.
The problem:
After performing in vivo electrophysiological recordings from visual areas of the mouse brain, we perform histology to validate things like the electrode insertion site (marked by lipophilic dyes), virus expression etc.. To do so, we fixate the brain using perfusion with PFA, and produce vibratome slices of ca. 50 mum thickness.
The question is how to achieve a perfectly coronal cut that allows one to reference the slices with common mouse brain atlases such as Franklin & Paxinos and the Allen Mouse Brain Atlas, and to see the entire electrode insertion tract?
One solution:
We now standardly immerse the brains in agarose which, once solidified, can be placed as a block onto the slicing stage. To do so, we place the brain upside-down (i.e. dorsal surface downwards to achieve a skull-flat configuration which aligns the bregma-lambda plane and is the configuration used in the aforementioned atlases.) into cell culture chambers (https://elabsaver.com/product/cell-culture-chamber-slides/), which allow us to align the brain more precisely in the agarose before it hardens. The cell culture chambers also allow us to easily extract the agarose block whose sides are in line with the coronal, horizontal and sagittal plane of the mouse brain.
Obviously, there is always some slight variability in the precise cutting plane, but each of these steps increases the level of precision. We are currently working on circumventing the problem by post-processing the microscope images, specifically by creating a 3D-stack of multiple brain slices and cutting through that stack virtually to produce any desired cutting plane. Furthermore, there are promising software packages for automated cell registration and atlas referencing, as found here: http://www.wholebrainsoftware.org/.
[ORIGINAL POST]
"Dear all,
my question is how to create one initial coronal cut of the mouse brain, for the purpose of creating a base through which the brain can be glued onto a base plate of a vibratome. My method so far has been to lay the brain with its dorsal surface downwards to achieve a skull-flat configuration which aligns the bregma-lambda plane and is the configuration used by brain atlases incl. Franklin & Paxinos and the Allen Mouse Brain Atlas. However, only using a hand-held razor-blade I am concerned about the obvious variability of the slicing plane. I would be glad to hear your suggestions on how to improve the method.
Many thanks!
Yannik"
If you have access to a tissue chopper, you could use that to block your brain in a more precise plane, as the blade is perfectly vertical (perpendicular to bregma-lambda plane)
As Alberto pointed, the use of a brain matrix/slicer will help. See link for examples
http://www.zivicinstruments.com/mouse-brain-slicers-matrix-section.html
Yannik, you don't say whether your brain has been fixed or is left native prior to cutting. You also didn't specify the needed thickness of your "perfect coronal sections". Alberto and David have given good practical hints how to overcome....
So - in the end depending on your task - you also could construct -build a device by your own ( thinking of a gauge/jig, which might be similar to a miniaturized "egg-cutter" ) as we have done such in the late 70ies. Best wishes and good luck, WM.
Thank you all very much for your answers indeed. The answers given by Alberto and David are exactly what I was looking for! As Wolfgang correctly pointed out, my question was not specific enough (my apologies), prompting me to change the question title and content.
Yannik,
I wish you all the best for doing your studies on mouse brain tissue slices. Your request brought up memories of my Dissertation Theme on developing nuclear regions in rat brain... finding a way for (almost perfect ) alignment of fixed brain without having a proper brain slicer matrix and/or ( a highly desired, but at that time =1978-1979 not financable) vibratome. If you have the time: I greatly should acknowledge your referencing the Franklin & Paxinos and the Allen Mouse Brain Atlas (for bibliographic purposes only). Thank you!
Yannik, could you tell me how did you prepared the agarose? Which concentration did you used? And did you diluted in PB or PBS? Thank you!
One can use the so called matrices for brain but they are not precise enough and are mostly dedicated to tox pathology. There the precision matters but not that much.
Anothe option is if you use dyes that arte fixable in formaldehyde, to perform brain clearing to see the positions. Aftewards you can "paint" the base of the cleared fixed brain with a surgical marker, fix it and do trims of the brain in the position of the electrodes. The surgical marker will be your orientation and will allow you to stitch the slices in image analysis solution if you need.
Afterwards, the brains are paraffin emedded and precise cut two serial semi-thin sections cections will allow to have the fixed dye and the stained brain.
Then you need a neuropathologist like Prof. Dr. Matiasek to analyze the structures.
Then you are good to go.
Cheers,
Ivan
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