Cool the Cryoblock & Heat Sinks
The cryoblock & heat sinks can be kept ready to use by housing them in an ultra low (-70C) freezer, or a dry ice storage container. The cryoblock & heat sinks can also be cooled from room temperature with dry ice slabs or pellets.
When housed in an ultra low freezer or dry ice storage container they can be retrieved, placed in the caddy and used immediately. With the lid on the caddy the cryoblock will remain cold enough to use for at least an hour. This is the most efficient cooling strategy when doing occasional frozen sections.
For extended and/or multi block frozen sections the cryoblock & heat sinks can be retrieved from cold storage and used immediately, with the temperature being maintained over the day with dry ice.
A room temperature block can be cooled to a usable temperature by placing a slab of dry ice on the block face or burying the block with dry ice pellets. It takes about fifteen minutes to reach -60C which is a usable temperature. A cooling time of thirty minutes is preferred but not necessary. Dry ice chunks or pellets in the space between the block and sloped sides of the caddy will maintain the temperature all day.
Align the Block Face and Cutting Plane
This method is capable of unprecedented accuracy, demonstrating a full face with almost no trimming in, however the accuracy can only be realized if the cutting plane of the microtome and chuck face are aligned. It is worth taking the time to establish this alignment, once done it should not need re-adjusting.
Try freezing some blank blocks ( with no specimen) and use them for the alignment. When the first contact with the knife trims off a full block face and not just a corner or side the desired alignment has been achieved.
Steps for Completion
- First label an appropriate sized mold, then add just enough gel to the make the inner base of the mold sticky, this will help hold the tissue in place.
- Position the specimen so that the surface of interest is face down in the mold, invert the mold and verify that the orientation is correct.
- If necessary adjust the orientation and re-verify, repeat this cycle until the specimen is adequately oriented.
- Scrape, brush, or wipe any frost buildup from the freezing platform of the cryoblock.
- Place the mold on the freezing platform, the thin film of gel freezes in seconds turning an opaque white and securing the specimen orientation.
- Over fill the mold with gel so that a meniscus is formed that is higher than the mold shoulders.
- When the base of the mold has frozen and the meniscus is still fluid place a labelled, room temperature chuck over the mold, gently press down so the chuck settles against the mold shoulders and any excess gel is expelled into the chuck channels. This settling of the chuck against the mold produces a viscous coupling which allows the chuck and mold to be lifted as one unit, and placed into a chuck stem hole. The large footprint of the chuck accelerates freezing of the gel, place a heat sinks on the mold face to prevent the block face from thawing and to accelerates freezing.
- As soon as freezing is complete remove the heat sink, then lift off and discard the mold.
- Place the chuck in a holder and transfer to the cryostat.
- Allow the chuck about 3 seconds to come up to cryostat temperature before starting to cut.
First label a room temperature chuck, then fill the hatched area to be used with gel, using only enough to fill the grooves and create a sticky surface. Place the chuck in a holder and it is ready to go. Once the tissue is mounted place the chuck in a chuck stem hole in the cryoblock and add additional gel for tissue support during cutting. If desired a heat sink can be used to accelerate freezing. As soon as the gel has frozen transfer the chuck to the cryostat chamber and let it come up to cutting temperature (about 30 seconds) before trimming in.