I dont have carboxygen, and I have tried placing the slices in kreb's buffer from this formula: https://health.uconn.edu/li-lab/wp-content/uploads/sites/129/2017/06/preparation_of_brain_slices.pdf
It works great if I keep the slice on ice the entire time.
I read in some article that magnesium based solution works best for slicing brains... but in other article, sucrose based solution works great. Which ones are better from your experience? I have only been using Kreb's buffer and I might follow what works better.
I see many of articles that its better to isolate brain and slice within approximately 10min, but is that realistic? I use hand slicer, and it takes me at least 3min to get back to lab so it takes 20-30minutes total from isolation to slices... Also, is it practical to weight brain slice? I have terrible time trying to weighing it to approximately same size... I feel like I'm not getting accurate measurement with solution on tissue. When I try to dry the slice with filter paper or KimTech, brain slice rips apart... Any ideas or methods you use that can be shared?
Thank you.
Dear Ilene,
Yes, you can remove and slice a single brain in 10 minutes or less.
I can only speak from the experience of using the TTC staining to look for post-ischemic damage in rat brain, so the brain slices only had to "live" during incubation in the tetrazolium salt solution. However, this is the procedure we came up with that allowed us to rapidly slice the brain and successfully perform staining and Western blot analysis on alternating slices: We had a room in the vivarium where we were allowed to sacrifice the animals. I had a stainless steel brain matrix that I used for hand slicing, and lots of clean double-edged razor blades. I would chill these in the fridge overnight, as well as several beakers of ACSF. These were all kept on ice in the procedure room. After sacrificing a single animal, the brain was quickly removed using a sharpened narrow weighing spatula to easily slice thru the cranial nerves (it takes practice, but two to four quick cuts with the appropriate sized rongeurs should expose most of the brain after the skin is opened over the midline) and placed in the beaker of ACSF for 5 minutes. This would firm up the brain to allow easy slicing. The brain was then put into the chilled matrix and the razor blades inserted to slice the brain. My biochemist partner and I then worked as a team, so that I would remove the first slice I needed and place into another beaker of ACSF, then give the next slice to him to dissect on a Parafilm sheet on ice. He would then put his brain bit into Laemmli buffer and quickly grind with a disposable micro pestle (He was very fast at dissections--seconds, really). By that time, I had the next two slices in the series removed from the matrix. Many times the slices adhered to the razor blades even though they were Teflon-coated, but that just made it easier to remove them from the matrix.
If you are using a McIlwain tissue chopper, you can also make slices rapidly. However, I have only used this on retina and hippocampus.
Whenever I have had to weigh fresh tissue, I have opted to use either Parafilm or heavy duty plastic wrap.
Good Luck!
Jill
Thank you Jill! I will try cooling all the utensil that will be used and work everything on ice. I am guessing slicing may be better where I dissect the brain? I have been doing it under the hood, but I think your method is more practical. Thank you again for the information!
Dear Ilene,
If you need sterile slices, then I would suggest that you sacrifice and dissect the brain close to your tissue culture hood. It depends on the setup of your lab, and what your IACUC-approved animal protocol allows. I have cultured several types of neurons, and close proximity to the hood always worked best. I have performed initial dissections using benchtop sterile technique (no hood, just rely on speed and sterile instruments like survival surgery). Then I rinsed the tissue several times in successive baths of sterile solutions (a new container for each rinse) in the hood. This helps dilute contaminants. Again, depending on your lab setup, it is good to do this in a hood that isn't used for critical long-term cultures. Or coordinate with lab mates so that they know that you will be working on primary tissues in the area.
Some institutions have facilities in their vivariums for sterile work, which would at least let you get the brain sliced and into the incubation buffer of your choice.
Good Luck!
Jill
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