reducing freezing artefact

[cs.vic]

Hi everyone,

Our  lab has been having ongoing problems with large holes in tissue cross-sections.  We believe this to be due to ice crystals formed during freezing, but nothing we seem to try to improve it is working.

We only deal with human skeletal muscle biopsies, and immediately after the biopsy is taken about 20-30mg of muscle is blotted dry mounted in OCT and snap frozen in isopentane cooled in liquid N2.  We thought that perhaps we weren't letting the isopentane cool enough so we purchased a digital thermometer, and now always ensure that the isopentane is at least -140degC or colder.  But we haven't seen any improvement of our sections.

I do cut them quite thin at 5-6um.  But even at 10um the quality is still extremely poor.

Would freezing in the isopentane without the OCT help?

Any suggestions would be greatly appreaciated, because as far as I can tell we are doing everything right but getting crappy results.

thanks.
cs

[cs.vic]

Has anyone compared results of freezing tissue in isopentane with and without OCT.  I have done a bit of reading and it seems some labs don't use OCT, and I have read that if you use to much then it can have an insulating effect and result in more artefact.  So I'm thinking maybe I'll try freezing some muscle straight in isopentane without the OCT and only add that when I go to section the samples.

Any thoughts?

[Frank]

Hi,
Maybe the reason is that the OCT isn't having enough time to fully penetrate in the tissue because of it's viscosity. I did some testing in the 90's on lung tissue and got the best results with a mixture of 10 % sucrose in PBS. When the tissue sinks in the solution (first it will float) then it is ready to be put in OCT. Afterwards freeze it in. The concentration of sucrose can be even 20-30 %. Sucrose is a well known cryoprotectant.
I hope this is a some help to you.
Frank

[Cardio]

Has anyone compared results of freezing tissue in isopentane with and without OCT.  I have done a bit of reading and it seems some labs don't use OCT, and I have read that if you use to much then it can have an insulating effect and result in more artefact.  So I'm thinking maybe I'll try freezing some muscle straight in isopentane without the OCT and only add that when I go to section the samples.

Any thoughts?

Well it does take longer if you have tissue embedded in OCT. OF course its also easier to cut the tissue if its embedded in OCT prior to freezing. The main reason you are getting artifacts is that you don't use a cryoprotection step ie sucrose in PBS. This removes the water from the tissue and limits freezing artifacts. THe thinner you cut the more artifacts you will see.
IF you add OCT to frozen tissue it doesn't always adhere around sections. You can end up with your tissue falling off during sectioning. You could section the tissue without embedding it OCT. Just take a dab of OCT on the pedestal to adhere the tissue and cut it that way. ITs harder but it can be done.