I am running immunohistochemistry on rat brain tissue, and the sections will be stained while free-floating. However, we need to reliably know the left from right hemisphere. We cannot take a notch out of the cortex (which we have done previously), because we are taking a full survey of the brain.
Could someone recommend a method to label the hemispheres? Is there some sort of marker or pen that is visible to the eye but would not affect staining?
- Thank you -
Hi Nathan,
Might not be of much help but I used to make a small puncture on one hemisphere using either a pushpin or a needle while I was sectioning. I had to repeat the puncture every so many slices as I didn't want to push too hard and make the hole too big, just big enough to see. Also, you could create your own pen using lipophilip dyes in a solvent and then infiltrate filter paper with that solution and let it dry. The filter paper will allow you to put very small amount of the lipophilic dye on the brain. Of course the lipophilic dye will diffuse and if you are doing fluorescence imaging it will take up one of your channels, so you'll need to consider and test that but you should be able to see it with your bare eyes. Maybe the idea would also work with another kind of dye that doesn't diffuse so much.
Good luck!
Depending on which area you are interested in (i.e. deeper structures), you could make a shallow longitudinal incision down one hemisphere before cutting - I did this when look at the amygdala and it's a very simple way to tell left from right!
You could put a suture loop through each slice from the same hemisphere.
You could use India ink or any other tissue-marking dye and mark the surface of one hemisphere. This would have to be done before embedding and sectioning.The dye can be seen microscopically even after staining.
you can embed the tissue in gelatin and make a notch in the gelatin.
Depending upon how the brain is fixed and what is embedded in (e.g., I note the first suggestion of gelatin embedding, which I have used very successfully), the easiest marker that does not disrupt the neuroanatomy is an incision on one of the cerebral hemispheres. Just remember to write it down and be consistent thereafter.
I would use a little dab of lipophilic dye, like DiI:
https://en.wikipedia.org/wiki/DiI
Are you planning to use fluorescent IHC? If you do, just make sure that the dye doesn't overlap with the emission profile of the fluorophore you will use!
Embed in egg yolk and make a notch in the yolk to mark the orientation and to distinguish left from right hemispheres.
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