Milestone PrestoCHILL, Operator'S Manual

Page: 50 / 72

PrestoCHILL MM138-003
– Operator Manual
50
A. Appendix
– Tips and Tricks
A.1. Fat tissues
Fatty tissues are difficult to section using a cryostat because they are non-aqueous materials and simply do
not freeze. The problem is clear when the fat begins to smear and impedes cutting of any tissue in its path.
To make fat hard enough to section, it must be cooled to a very low temperature that often is too cold for
non-fatty components present in the same sample. Another problem is that fat does not adhere well to the
embedding medium.
Milestone studied a new protocol that allows fat cutting.
1) Prepare the mould by cleaning with paper soaked in alcohol and placing the appropriate chuck on it
(this procedure will prevent ice inside the mould).
Cleaning of the mould is very important to avoid the block from sticking in the mould.
2) Prepare the sample on the spatula with thin layer of MCC medium under it. If preferable, it is
possible to use the paper disk method (see BOP 102).
3) Transfer the sample inside the mould and immediately fill the mould with MCC medium.
4) Put chuck and heat extractor.
5) Freeze for 4 minutes at -40°C.
6) Trim the block in the cryostat and try to get a thin slice. It is possible that during the trimming the
block temperature became too warm to section.
7) Put a mark on the block in order to recognize the position of the block inside the cryostat.
8) Cool down the block inside the PrestoCHILL for 1 minute. Be sure to put the block surface in contact
with the PrestoCHILL cold plate: not in the mould used for the freezing because the thickness of the
block now is reduced, it is better to use a larger mould or a free space on the metal plate.