Hi, I'm an undergraduate student working on a thesis project. I am sectioning rat plantaris muscle in a cryostat and measuring area (hypertrophy/atrophy) comapred to other subjects, as well as counting the number of muscle fibres. A graduate student excised and froze the tissue in OCT, and it is my job to section, stain, and analyze the muscle samples in the lab. I've noticed that no matter how good or bad my sections are, there are always numerous folds in the tissues. I'm not sure whether or not this has to do with my sectioning, my staining or the freezing process.
The stain I was taught is quite simple: samples are left for ~30 s in 0.1% solution of Toludine blue, then dipped in deionized water 3 times, are dried, and then can be looked at under a light microscope.
The graduate student who froze these tissues is gone and I cannot reach him. My fellow lab mates do other immunohistochemistry work and have no experience with what I am doing. Any information is helpful!
Dear Alex,
The problem might be that you froze your muscle tissue too slowly and if so, it can lead to freezing artifacts and "holes" in your sections. You should keep the muscle straight with the appropriate forceps and freeze it in isopentane mounted on a small cork and gel. The section needs to be perpendicular to the length of the fascicles. There should be videos online illustrating the technique. Cutting the sections is a tedious work and freezing is a crucial step for not repeating the steps multiple times. you could also consult dpt. of pathology doing muscle bx preparation and staining in your area.
I hope it helps,
Best,
Giorgio
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