Solvent-free tissue processing using supercritical carbon dioxide

Xylene is most often employed in tissue processing protocols for paraffin embedding, but poses a health hazard. The aim of this study was to evaluate a solvent-free processing protocol that uses supercritical carbon dioxide (scCO(2) ) as an intermediate.

A series of tests (with bovine tissues) was run, evaluating dehydration and tissue shrinkage in our new scCO(2) -based protocol as compared with routine processing using a graded ethanol and xylene series. A series of tests was then run to evaluate the significance of processing parameters for the outcome. Finally, a validation series was performed with optimal conditions, testing various human tissues with several staining methods. The tissue water content after paraffination was the same with our new scCO(2) -based protocol and the routine xylene-based protocol. Tissue shrinkage was similar with the two methods, at ∼15%, which is also similar to values in the literature. In the validation series, the human tissues showed good morphology with strong staining, probably because of stronger antigenicity.

This scCO(2) -based protocol has been shown to be a good solvent-free, alternative form of tissue processing. Although not the focus of this article, the time needed for tissue processing with this new protocol is within 4 h, and there is no need to change macroscopy/sectioning protocols.