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Post subject: Gastrocnemius-plantaris complex muscle sectioning problem
Greetings to all,
Lately i have been trying to get good sections of the gastrcnemius-plantaris complex.
The problem is, since the muscle is composed of lateral gastroc, medial gastroc and plantaris, while sectioning, these muscle tends to separate. As a result, in the obtained section, i have a Y-shaped crack separating the whole muscle - complex in to 3 parts.
Please provide some advice/suggestions or tricks to get good sections of big muscle like this.
Animal= Rat
Muscle is covered with OCT, and flash-frozen in pre-cooled isopentane in liquid N2. No fixation is done prior to freezing.
Section Thickness = 10µm
Cryostat temperature = 20 - 22C
Hope to hear from ppl soon!
BEST,
AR
Thu Nov 03, 2011 3:49 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: ppl reply....
Hey, AR...you getting v. good in "succinctness"
( a lot of info in few words)
Yes, that complex of muscles is not easy...
A very careful dissection is MOST important.
After that CAREFUL dissection, I place that muscle complex on a double piece of tin foil that I have smeared with OCT.
I carefully arrange the muscle ( sure, give it some nudges to try to re-arrange it like it was in "real life") and then...leave it for a short time ( ~1 minute)so it can relax ( remember that muscle has a Tonus that will react to dissection).
I then apply more OCT over the top of the muscle.
Then, I turn up one corner of the tin foil so I can lift up the foil into N2-cooled iso-Pentane, to freeze.
Sure, this means that there is very little OCT around the muscle, so cryosectioning must be GOOD.
For this paper...many , many muscles were dissected out and frozen:
http://jcb.rupress.org/content/135/1/241.full.pdf
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