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.