Embedding media for cryomicrotomy: an applicative reappraisal.

We reassess here the formulation of cryoembedding media in connection with recent developments in commercial cryomicrotomes. Water-based solutions of polyvinyl alcohols were our starting media, and each of 2 different polymers (56-98, MW approximately 195000; and 6-98, MW approximately 47000) showed a critical concentration for optimum sectioning. At higher or lower polymer concentrations, wrinkles and folds became apparent in tissue areas of sections, or in the sectioned embedding medium areas between tissues, respectively. Addition of polyethylene glycol (MW 380-420) further facilitated and improved sectioning, resulting in frozen tissue blocks that cut well in the 2 to 100 microm range and further, using disposable blades throughout. Applying a wide temperature differential between tissue specimen (-11 degrees C to -13 degrees C) and cutting knife (-33 degrees C to -35 degrees C), serial adjacent sections were reproducibly obtained at a 2-microm setting, singly or in short ribbons. Embedding media of low and high viscosity were obtained, depending on the polyvinyl alcohol polymer used, and could be applied sequentially for tissue infiltration followed by embedding with precise sample orientation. When required, media were made semisolid by addition of carboxymethylcellulose.

Equipment testing and tuning: the cold-knife cryomicrotome microm HM-560.

A major innovative feature of the Microm HM-560 cryomicrotome is the independent control of specimen and knife temperatures. We used such equipment with a variety of tissues, and fixation and freezing procedures. High-quality sectioning was reproducibly obtained using 1) a low temperature setting for the sectioning blade ("cold knife," about -33 degrees C); 2) a comparatively high temperature for the specimen; and 3) a suitable mounting medium, which would remain solid up to about 0 degrees C. Specimen temperature was set between -8 degrees and -15 degrees C for 4-microm sectioning, higher temperatures (-1 degrees to -8 degrees C) being appropriate when cutting at 10 to 20 microm. Under such conditions, disposable blades were effective throughout, while a modified antiroll plate profile further enhanced usability. After intensive use for almost 3 years, by more than 15 different users, the cryomicrotome is in excellent working order.