I wash rat heart (left ventricle) 2 times with PBS over an ice bath, dry it with paper towels and then embed it in OCT over cooling bath. Lately, while sectioning in cryostat tissue is falling apart, creating a hole in the center of the OCT film.
What could be the reasons behind this and how to prevent this in order to get good cryosections? Please help and share your experience and expertise with me.
2 reasons could be responsible for that probelm. 1. The slides that you use. I recommand to use superfrost plus slides. 2. this could happen when you dry the sections in the cryostate after you have transfered them onto the slides. The sections have to melt on the slide. After you have transfered them let them dry outside the cryostate for an hour. Then deep freeze them in the -80°C fridge until you will use them for immunohistochemistry or ISH.
Good luck!
After mounting the tissue on the slide (I also recommend superfrost plus), I warm the slide (37C for 30-60 minutes) to firmly attach the tissue to the slide. This has not caused me any staining issues (fluoro or in situ) and, although I typically section brain tissue, the same principles apply.
Yes, I use superfrost plus glass slides for my sections. There is no problem in getting tissue onto slides. But, my problem is while cutting in cyrostat, tissue falls separate making a hole in OCT, it doesn't come out as a film together.
That is normal. The OCT will eventually be washed away, so no loss there. It does provide more to work with when mounting but it is also common practice with brain tissue to take your section and remove the excess OCT prior to mounting the brain tissue onto the slide. If, however, you want to keep the two together more, I recommend the following: prior to freezing the heart in OCT, let is equilibrate in room temp OCT for 10 minutes or so, then move to the tissue mould and add fresh OCT for freezing.
Usually if we use fresh tissue, we directly embed into OCT then isopentan alc. cooled by dry ice, and keep in -80 till cut, or if use formalin-fixed samples, it should be kept in 10% then 30% sucrose till sink then OCT and -80C. before putting in OCT, put on a fiberless paper to remove extra solutions. always keep OCT blocks in the cryostat chamber for about 30 min so the tissue and OCT get same cryostat temperature.
I hope this is helpful.
I do not embed the tissue completly in OCT. I use OCT only to fix the tissue block on the tissue table of the cryostate. This makes the mounting of the sections much easier. If you need some embedding to keep different tissue layers together I recommand agar or gelatine.
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