I'm having difficulty cutting fixed and dissected rat hippocampus tissue
with a cryostat. The slices come out with closely spaced cuts and
tears parallel to the blade and are too damaged to be useful. I've tried:
1) cutting at different blade angles
2) different temperatures from -5 to -25
3) three different blades all freshly sharpened
4) cryoprotecting in 15% or 30% sucrose
5) with or without using an anti-roll guide
6) different slice thickness 15 to 40 microns
In another lab I've used a cryostat for three years and never had any
problems but this one has me stumped. I've asked other people and have
tried all their suggestions with nothing helping. I've wondered about
the fixative though others have said it wouldn't make a difference. I'm
using an ethanol/acetic acid/chloroform mixture which works great for my
immunocytochemistry. Can the fixative and/or fixation time be making the
tissue more likely to be shredded while cutting? I've fixed for 4, 6
hours and overnight, with the tissue coming out shredded each time.
I thought it was a mechanical problem but it turns out it's not something
as simple as using a sharper blade. I've also replaced our anti-roll guide.
Any experienced cryostat users out there who know what's going on here?
Brian Scott | "In other studies you go as far as others have gone
brians at interlog.com | before you, and there is nothing more to know; but
M.Sc. student in | in scientific pursuit there is continual food for
Neurophysiology | discovery and wonder." - Victor Frankenstein