Hi everyone
Im trying to slice my mice brains with the cryostat, however, I haven't been successful, the tissue seems to stretch and then it breaks.
Let me tell you the story about these brains:
These brains were perfused with 4% PAF and Gadoteridol for MRI, we did the MRIs and then kept these brains in the fridge (4ºC) in PBS and Sodium Azide for 6 months aprox. A week ago I transferred them to 10% sucrose in PBS (1x) and then 30% sucrose in PBS (1x) and today I tried to cut them, the cryostat is working fine, I sliced other brains before these ones, however the tissue of these brains breaks, and I don't know why.
I searched in the literature and found 2 papers that did these same (MRI + sectioning + IHC) things BUT they embedded their samples in paraffin, and my question is, is there a specific reason to use paraffin embedded tissue in the microtome instead of frozen tissue with OCT in the cryostat with the conditions I mentioned before.
I attached a video, I don't usually cut this slow it was only meant to focus the video.
PD. The glass doesn't have any nicks and the blade is new. Any other ideas?
Thanks in advance!!!
there may be nothing wrong, change the blade or move it left or right, there might be a notch on the blade, which grabs the tissue and makes it shrink. I've seen this. Maybe this particular sample was not perfused well and there is blood in the brain (different density); drop the temperature to -22 -24C and try again, keep the sash as closed as possible to keep the temp inside low. If nothing works -you can take a brush, gently place section on the slide with 1mp PBS on it and then carefully remove PBS from underneath the section. I've done tons of frozen sections and used them for floating IHC (stain, then mount), so you can use the same logistics. General comment - the samples were stored for too long, not sure about the stability of epitopes. Good luck
Thank you for your answer, I'll work on those suggestions. Wish me luck for tomorrow ❤️
Hi Marcela,
My limited understanding of neural tissue is that you want to fix it before you perform OCT embedding and frozen sectioning, or you will not get publication-worthy images. To do this, the sample must be fixed, then treated to a 30% sucrose solution (in PBS) for a few days before it is then OCT-embedded and microtomed.
Hi Marcela,
I don't know if your problem with section cutting is over after lowering the cryostat temperature and changing the blade. After watching the video, it looks like the anti-roll plate might be the problem. Clean it or change it if it has a nick.
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