▲ 1 r/labrats
Hey labrats! I’m struggling with sectioning 40 µm slices of PBS-perfused, 4% PFA post-fixed mouse brains on a Leica VT 1200S vibratome, and I could really use some advice.
The problem: I need to cut whole brains (coronal sections), so I embed them in 4% agarose (made with PBS) to create an agarose mold. But no matter what I try, I keep getting one good slice, then one bad slice—where the "bad" slice is either damaged, torn, or missing entirely and the good slice seems over 40 microns of width. I suspect my agarose might be the culprit (too soft? bubbles?), but I’m not sure how to fix it.
What I’ve tried so far:
- Using 3% or 4% agarose
- Adjusting vibratome speed and amplitude
- Checking blade sharpness
- Checking that the brains are well glued
So if anyone has a good protocol or else, any suggestions would be hugely appreciated! Thanks in advance !
u/AlphazZ9 — 7 days ago