Hi there,
so I am working with a frozen microtome to cut murine spinal cord sections, but none of my sections have consistent white matter boarders. They are basically either frayed or wobbly, so volume analysis is basically impossible. I have tried and keep the sections colder but that leads to them tearing. Any ideas what I could try to get rid of this problem?
Thanks!
And what about the temperature? Have you tried lower or higher temps.?
Hi Charlott it sounds a lot like a blade problem. Do you change your microtome blade often? Sometimes "grit" or particulates accumulate on the blade which can lead to fraying of the section. Also if the blade is slightly loose it can cause "wobbling" or "wavey" sections.
The first thing I would try is replacing the blade and make sure it is tight.
If the tissue is well fixed you shold find the optimum temperature during sectioning. If the block is too cold, it breaks, if soft you may have the problem you mentioned. I agree with Gary regarding tha blade. Also the angle of the blade to the horizontal and the longitudinal plane should be correctly adjusted. The appropriate speed of moving the blade is also important. Yo shuld play with this factors until the sections will be acceptable.
I guess the Problem is from preservation, possibly your Fixative had alot of water than its chemical component. Therefore, the marginal Areas of your tissue will be oedematous. I mean it will soak water from the Fixative. Check the Fixative to be sure of the concentrations. If also you had use fresh tissues, then necrosis have set in meaning they were processed after more than 30 minutes of salvaging the animal.
Good luck.
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