Ø Before starting the muscle biopsy procedure, pour 100 mL (approximately 1/3 of beaker) isopentane (2-methybutane) to a glass beaker or a steel beaker.
Ø Suspend the beaker in a bath of liquid nitrogen (either in a larger beaker or Styrofoam box) and leave until the isopentane ice crystals start to form on the bottom of the beaker and on the sides of the beaker. When this occurs, isopentane has reached optimal freezing temperature (-150 °C). Time taken averages between 3-5 minutes.
Ø Following the skeletal muscle biopsy, remove the excess moisture by dabbing the tissue on the filter paper. Place muscle in proper orientation. Cover it in OCT (Optimum Cutting Temperature compound), and lay it flat on a labeled disposable freezing mold. Freeze the muscle for about 10-12 seconds (Being very small muscle tissue)
Ø Transfer the frozen tissue on to dry ice and let the isopentane evaporate( approx. 20minutes) and wrap the tissues in aluminum foil and store them temperature (-70 °C to -80 °C) .
I have done this a few thousand times. Your procedure should work. I might do a couple things differently. 100 ml of isopentane is more than I would use. It will take a while for the isopentane to cool down. However, if you are willing to wait, you can use 100 ml. The most important step, I believe, to getting artifact-free sections is to cool the muscle specimen/OCT as quickly as possible. I will plunge the specimen/OCT to the bottom of the cooled isopentane as fast as possible. I also like to keep the mass of specimen/OCT small (e.g., maybe
At step 2, instead of liquid nitrogen we use dry ice to freeze the tissue in OCT.
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