Author Topic: Trouble Sectioning mouse skeletal muscle.  (Read 1743 times)

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Offline VShemica

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Trouble Sectioning mouse skeletal muscle.
« on: December 16, 2014, 04:38:05 PM »
Hello everyone,

I do not have much experience sectioning muscle tissue and I am having a troublesome time sectioning FFPE mouse skeletal muscle at 5 microns that's to be used for IHC.  The problems I'm running into are:

A) Excessive knife marks (I'm using a fresh portion of blade every 1-2 sections)
B) What looks like veneican blinds/cracking effects only around the edges of the tissue (this is after soaking in a ice water bath for long periods of time)

Some background:

A) Fixation - the researcher placed  tissue in 10% NBF for @ 1 month max prior to handing it off to me
B) Grossing - I dissected the muscle specimen into @ 6 x 3 x 2 mm pieces for processing
C) Processing -    1) 10% NBF                  10mins  40oC
                             2) 80% Ethanol            20mins  40oC
                             3) 95% Ethanol            20mins  40oC
                             4) 100% Ethanol (x3)   20mins  40oC
                             5) Xylene (x2)               20mins  40oC
                             6) Paraffin (x4)             20mins   58oC

I'd love to hear from some of you histotechs out there who has had hands on experience with muscle tissue as to any solutions for my current case and also anything that can be done in the future to prevent these artifacts.

Thanks in advance,

Mica
« Last Edit: December 16, 2014, 04:49:17 PM by VShemica »

Trouble Sectioning mouse skeletal muscle.
« on: December 16, 2014, 04:38:05 PM »

Offline gula

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Re: Trouble Sectioning mouse skeletal muscle.
« Reply #1 on: December 17, 2014, 10:31:18 AM »
This sounds like dirty paraffin. Dirt-particles can lead to knife marks. It is recognisible, if the marks are also in the "only-paraffin-part" of  the block.  You can try to filter the paraffin. Melt the block again and embed in clean paraffin.
If the tissue itself has hard particles inside, it may be calcification. Is that possible in your case? broken parts of rat-bone?

Venecian blind is usually caused by some loose part of the microtome. Knifeholder, blockholder, ...
or too fast cutting, vibrations of the knife.
Some tissue is sensible for too long soaking. This can be seen, if the tissue "swells" out of the block-surface.
gula

Offline VShemica

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Re: Trouble Sectioning mouse skeletal muscle.
« Reply #2 on: January 06, 2015, 04:45:32 PM »
Thank you gula for the info.  The muscle was skeletal muscle, but no bones fragments were present during grossing that I noticed.  So I will give changing the paraffin a try.  I never even thought of that.  Also, I will make sure my adjustable microtome parts are thighten.  Thanks again!
« Last Edit: January 06, 2015, 04:48:57 PM by VShemica »

Re: Trouble Sectioning mouse skeletal muscle.
« Reply #2 on: January 06, 2015, 04:45:32 PM »