Muscle Cross Sectioning Help - Samples look like they have tiny holes in them (Apr/27/2011 )
I am using a cryostat to take cross sections of skeletal muscle. Cutting temp = -25C, thickness = 10-12micrometers, no problem with samples sticking to the role plate or anything like that. With in the sample, some fibers look great and some look like there are a bunch of tiny holes in them. With the fibers that have the holes in them, their cell membranes are usually smudged and its hard to distinguish between fibers.
There are also a few tears that seem to happen in the same place for every cross section I take.
If anyone could help me out and tell me what is causing those fibers to have tiny holes in them I'd greatly appreciate it!
Thanks,
-Lyle
LyleBabcock on Wed Apr 27 16:41:51 2011 said:
I am using a cryostat to take cross sections of skeletal muscle. Cutting temp = -25C, thickness = 10-12micrometers, no problem with samples sticking to the role plate or anything like that. With in the sample, some fibers look great and some look like there are a bunch of tiny holes in them. With the fibers that have the holes in them, their cell membranes are usually smudged and its hard to distinguish between fibers.
There are also a few tears that seem to happen in the same place for every cross section I take.
If anyone could help me out and tell me what is causing those fibers to have tiny holes in them I'd greatly appreciate it!
Thanks,
-Lyle
Yes, I had the same problem and it was driving me insane. Now that I am already insane I stopped encasing my tissue in OCT and just attached the already frozen muscle piece to a cork plate by using a small drop of OCT... This way the muscle never thaws even a little. The holes you see are caused by freeze-damage which happens when your tissue is allowed to increase in temperature too much. How do you freeze your tissue after the dissection?
I am also currently cryosectioning some skeletal muscle sections and i am gettting some horrendous results with lots of holes in my samples presumably from ice crystal damage. The samples i am currently working on i did not freeze down myself however i asked the people who did freeze them down and the protocol they followed seems pretty standard ie freeze samples in liquid nitrogen cooled isopentane and then store at -80 till use. they did also cover the samples in oct before freezing down so maybe this has a negative effect. Is there a certain time frame the sample should be left in isopentane for?
However one thing that i would be interested to hear peoples thoughts on is wold the cryostat have any effect on the formation of the many muscle tears i see in my muscle sections persumably caused by ice crystal formation. I have recently moved to a new lab and the cryostat that we have here is one i dont like its a small box shaped one which can fit onto a table top and so i worry about how good its temperature regulation is as when you take off the lid the sample is quite close to the "outside" ie room temp. In my previous lab we had a bigger cyrostat with a sliding door and the sample was mounted a larger distance from the "outside" of the cryostat.
A second thing i was wondering about is the air drying of the sample. Once i cut my tissue section and have it on my slide can i leave that slide then straight away at room temperature for the sample to air dry and how long should i do this for? I have typically been leaving the sample for 10-15mins at room temp post cutting and then fixing in 2% paraformaldehyde.