In our lab, we freeze tissue samples from mice (embedded in OCT); we used to have a few problems with this method, using the standard plastic 15 mm cryomolds (frozen with dry ice & alocohol; or sometimes liquid nitrogen instead) -
1) It was very time-consuming. We would hold each individual cryomold over a beaker for several minutes - not only does this require a steady hand, it's slow and doesn't allow for multi-tasking. We freeze several samples a day, so it was taking up a lot of our time.
2) The frozen blocks can be difficult to extract from the mold.
3) Not enough space for labeling samples on the standard plastic cryomold.
4) Difficult to store samples in the mold; we would need to cover each with tinfoil before storing in the freezer (not optimal for protection of the frozen samples).
5) Samples would dry out in the freezer; we would need to wrap them with saran wrap to better preserve the mold prior to cutting.
I'm curious to see if anyone is having the same problems, and if there are any suggestions to improve the process?
For our own solution, we created a four-mold cryotray + box with platform - it lets us freeze four samples at the same time, hands-free. Each mold also has a lid for better protection/storage/hydration retention; and more space for labeling than a typical mold (see: www.sealnfreeze.com).
We do routinely these procedures. I fix tissue samples in 4% PFA, and go thru 15% and 30% sucrose, rinse in OCT two times and place tissue at the bottom of mold. And fill the mold with OCT,
Place the whole mold on flat surface of Dry ICE, it will freeze in 15-20 minutes or more. I take blocks of flat surfaced dry ice pieces. or crush dry ice to make surface flat. If you need to orient the tissue, you can hold the tissue with needle or fine foreceps in the right orientation while in mold is the beginning on dry ice.
You can store them in -70C. I never use alcohol to freeze blocks.
In cryostat, you directly place the inverted block on cold chuck (minus 22C), the block will attach to chuck, than remove the plastic mold or you can cut with scalpel if needed. I have never had problem in removing mold. If these molds are not good, change the company, ask for some samples, they will send.
Good luck
You can store the tissue in cryo vial or plain bottle. They can withstand extremely low temperature
Hi
Don't use alcohol use isopentane. If you lower the OCT/tissue sample into alcohol it can make your OCT "sticky". If you lower the OCT/tissue sample into isopentane well chilled with dry ice. It will snap freeze much faster and not be "sticky" afterwards. You can also use the stainless steel paraffin embedding moulds. The frozen OCT block is easy to remove from the stainless moulds. Any OCT block will freeze dry eventually, so keep them well wrapped up in foil.. I incorperate a labelled coloured "post it" type page marker, into my OCT block before it is frozen. That way when it is attached to the objective/specimen mount on the cryostat it is already labelled. It stops sample transcription error in one step....
I also allow my OCT to "meld" with the tissue for a short while, before I snap freeze. I will not freeze a lobe of mouse lung or liver down whole. I cut a 3 mm section out of the lobe, that way the OCT can ouze into the paranchyma of the tissue. A cuboid shaped tissue will freeze faster than a cubed tissue or OCT block.
I personally don't like the 30% sucrose method, I dont think they section well in the cryostat. Unless you turn the temperature down to -30C, it is like trying to section a "lollypop". All techniques have artefacts. For cryotomy the key is, to minimise them by RAPID freezing..
- Badges
- Science topic