Skeletal Muscle Cross Sectioning Help - Samples look like there are tiny holes in the fibers (Apr/26/2011 )
I am using a cryostat to take cross sections of skeletal muscle. Cutting temp = -25C, thickness = 10-12micrometers, no problem with samples sticking to the role plate or anything like that. With in the sample, some fibers look great and some look like there are a bunch of tiny holes in them. With the fibers that have the holes in them, their cell membranes are usually smudges and its hard to distinguish between fibers.
There are also a few tears that seem to happen in the same place for every cross section I take.
If anyone could help me out and tell me what is causing those fibers to have tiny holes in them I'd greatly appreciate it!
Thanks,
-Lyle
Maybe post this question in the histology section?
How old are those mouse? You would expect different morphology at different ages.
Rsm on Thu Apr 28 08:29:21 2011 said:
How old are those mouse? You would expect different morphology at different ages.
They're actually human muscle samples from the vastus lateralis, subject about 23 years old if that makes any difference.
let me preface this comment with: i have no experience with tissue sectioning.
that being said, wouldn't tearing occur if the tissue is not properly fixed (in this case, frozen)? are you sure that your tissue samples were completely frozen prior to cutting?
mdfenko on Thu Apr 28 14:15:43 2011 said:
let me preface this comment with: i have no experience with tissue sectioning.
that being said, wouldn't tearing occur if the tissue is not properly fixed (in this case, frozen)? are you sure that your tissue samples were completely frozen prior to cutting?
After mounting the samples on the cork, we froze them in isopentane for about 30sec-1min (-20C), then put them in liquid nitrogen and stored them in -80C until use.
Were the samples cryoprotected at all? It's possible ice crystal formation within the tissue may have caused problems during the freezing process.
gfischer on Thu Apr 28 18:33:28 2011 said:
Were the samples cryoprotected at all? It's possible ice crystal formation within the tissue may have caused problems during the freezing process.
Thats actually what I was thinking. I know you freeze it in isopentane at -20C first to avoid larger ice crystals forming and avoid freeze artifacts. They were not cryoprotected in any way other than using isopentane first like I just described. Is there a particular way to do this?
You can dehydrate using 20% Sucrose at 4C O/N, that's what I always do.