We have been working with muscle samples frozen with isopentane in liquid nitrogen for many years now. When we are going to cut them at the cryosection, we leave them for 20-30 min in -20o C, then we just remove the external part (we clean them as much as possible). The more you clean them, the easier it will be to cut them. However, when you are putting them in to the cryosector you have to cut them very slowly. But you shouldn't have any problem with this.

For my part, I work in a chamber at -30 ° C, with the temperature of the object set at -28 ° C. On the other hand, you should not hesitate to previously cut OCT block before cutting the muscle. If there are too many OCT, this will result in micro-vibrations, so the muscle will be damage.

Hi Peter,

it seems UR performing well, I do not really see which step of your protocol you could improve.... I just call attention to the fact that the way of freezing is very important. Are you taking care to freeze the samples very slowly and carefully? I have cryosectioned lots of OCT-embedded skeletal muscle tissue specimens (stored at -80 °C until sectioning) into 5-7 µm slices and there never has been a similar problem.

Hope you'll finally work it out,

Susanne

I agree with Spyridon..You need to remove as much of the OCT as possible before cutting. In my experience it is not optimal to freeze muscle in OCT, especially human muscle. Let the specimen equilibrate in the cryostat, then remove the OCT with a razor blade. In addition, I would try a new cutting blade, or move the blade to a new unused section. Good luck!

thanks everybody for yer answers to date. The suggestions to cut away as much as the oct as possible is something which i was thinking about as well indeed anita i personally prefer not to cover the sample in oct when freezing it down so it is good that you mention your experience with this as well. i have also tried new blades and different regions of the blade but still had the same problem as soon as the blade got in contact with the muscle tissue it struggled to cut through it.The blades we use are from leica and so i do not believe it is a quality issue with the blades as they work fine on the non oct covered muscle samples

As regards your point susanne i personally did not freeze down these samples but the protocol that was followed by the people doing the freezing down seems fine. Do you have any tips or little logistical suggestions on how you freeze down your samples? as we will be gettting more biopsy material in the next few months and i wold like to have the freeze down process optimised before then.

Interesintg nicolas that you work at a lower temp and still your samples are fine to section. I am very confused as to why the samples im sectioning are behaving in the way they are because from all of yer responses with regards yer experience of the sectioning muscle tissue ye have not encountered such an issue. I think i will also try and leave the samples in the cryostat for longer maybe 4-5hrs before cutting to see if this can maybe help "soften" the tissue for smoother sectioning.

Hi peter, as others suggest, you need to get rid of as much OCT as possible before cutting, but while keeping your samples frozen! Re alternative methods, the best samples Ihave seen are those frozen with extensive agitation directly in liquid nitrogen - this prevents ice crystal formation. May I suggest you contact Hans, he is of course an expert in this area! Hope all is well otherwise! Claire

For Skeletal muscles cryosectioning...

1. thinner sections around 4-6 um are easy to cut in cryostat

2.As mentioned above ideally limit the OCT surrounding the muscle sample as less as possible.

3. I was even had successful sections at -30 -32 oC cryostate temperatures

4. Important: briefly blow 1 or 2 gentle puffs of air by mouth on the top of the cutting surface of the tissue. This makes sections less brittle and more pliable and give you perfect cuts( time it exactly while you cut- need some practice- initially try this method some non important tissues)

Please always use face mask while cut the sections- this will help prevent in warm air from your nostrils being pumped into the cutting chamber. fluctuating cutting chamber temperatures makes tissues act unpredictably while you cut

Is it the same type of muscle (embedded and not in oct) that you have cut? I used to cut human muscle tissue and I have observed difference between samples depending on a type of disorder. Some of them were very difficult to cut due to tissue changes. I agree the less oct the better and also during freezing because it may cause artifacts. It is also important not to let the tissue dry out.

thanks for the suggestions claire and vidyasagar there defiently is universal agreement with the cutting away excess oct.

thank you for you comments justyna I was wondering what you mean by not letting the tissue dry out could you please explain as this could be someting important which our group needs to consider in the future

also justyna yes it is the same type of muscle in embedded and not in oct. Muscle samples were took from healthy 20 something males post exercise

I mean during storage in -80, it is good to use oct to protect tissue but not too much. Similarly when you cut the same sample several times use a little bit of oct to cover tissue before putting back to freezer but do not allow sample to thaw (too much of oct will result in melting tissue). Do not allow sample to dry out in cyrochamber as well, remove oct just before cutting.

Good luck!:)

Hi Peter, We freeze our human muscle in liquid nitrogen cooled isopentane. Never liquid nitrogen..the freeze damage is too extensive in our experience. We actually cool the isopentane till its just starting to freeze over on the bottom before we drop the muscle in. It's important to get good quality isopentane with low water content...And dont reuse the isopentane too much. Colleagues of ours actually mount the muscle on a small strip of card, as human muscle can be quite soft (depending on what muscle is biopsied and from whom it's from, age, lipid content etc etc) and plunge the sample into the cooled isopentane by holding the card with tweezers. That way the muscle is always orientated correctly and doesn't deform in an undesirable manner on freezing. I havnt done this myself, but it seems to work well for them. I dont have too much trouble just freezing the muscle by itself but It might be somthing to try if you are having issues. Just before freezing, carefully blot moisture from the muscle with some fine grade tissue to prevent ice crystal formation on the surface of the muscle then immediately freeze. We leave the sample in there for 10 to 20 seconds before removing to a cryovial in dry ice. As Clare mentioned, agitation by swirling the isopentane gently seems to help. remove sample with isopentane cooled tweezers. Cryovials are best for long term storage rather than standard laboratory tubes..so specimens don't dry out in the freezer -80oC. Separate tweezers for handling frozen and unfrozen muscle too is always handy! For sectioning, we mount on the chuck with minimal OCT. the chamber and chuck are cooled between -20oC to -25oC. We cut sections between 5 to 8 microns. Use a fine brush to remove any ice crystal formation on the blade (from breath). We mount on super frost plus slides.

Hi Peter. Hope you are well. I have used OCT for the 3D skeletal muscle cell cultures and because they have a large ratio of collagen to muscle, they are very difficult to section. I have been told by a reliable source (Dr. Georgina Ellison) that paraffin embedding will maintain the integrity of the tissue better than freezing, so when you come to section it doesn't fray.