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

I never had problem with this aproach

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..

Hi Dmitry:

I am not very sure about the 'dry out' problem in your experiment. But if it means many 'Swiss-cheese holes' inside your stained tissue slices, they were not caused by tissue drying in the fridge, but caused by ice crystals formed during your freezing process. To prevent this problem, tissues must be frozen as rapidly as possible. The EtOH-Dry ice bath did not provide a snap frozen environment (it's only ~-80 degree), and embedding tissues in big OCT chunk further slowed down your freezing speed (OCT is highly resistant to temp changes in surrounding environment), that's why you felt your freezing process was very slow.

My suggestions: 1. Use liquid nitrogen cooled isopentane to freeze tissues instead of EtOH-Dry ice bath. It would make your tissue frozen very rapidly and prevent forming big ice chunks inside tissue. 2. Do not embed tissues in OCT when you freeze tissue, but put tissues into liquid nitrogen pre-cooled isopentane directly. You need to determine the freezing time empirically: large tissues require more time to freeze but too much time in isopentane would cause tissue crack into pieces. You can store tissues in -80 fridge for short time period and embed tissues in OCT later. 3. When you embed tissues in OCT, set the temperature of

freezing microtome chamber to -6 to -8 degree (the freezing point of OCT in -10 degree) and use this temperature to pre-cool the OCT (Otherwise the room temperature OCT would thaw tissues and the ice crystals would be formed again by slow re-frezzing process). You also need to embed tissues inside the cold freezing microtome chamber. After embedding your tissues, transfer OCT chunks into a foam box containing dry ice to make them frozen completely.

You can also check a paper on JOVE (PMID: 4215994), which described freezing and cutting skeletal muscle tissues using this method in detail. But you may need to make some modifications based on the nature of the tissues you are studying.

Another option is fix the the tissues by formalin first then dehydrate tissues by highly concentrated sucrose before freezing tissues. In this method, you can embed tissues by OCT and freeze tissue using EtOH-dry ice bath. But you need to do antigen retrieval if you want to do IH or IF (different primary antibodies may need different optimal antigen retrieval methods) using those tissues. My friend in is another lab is handling tissues in this way but I don't know many technical details about it.

Hope it helps!