Investigations on ultrathin cryosections from cryoprotected and non-cryoprotected tissues by electron energy loss spectroscopy: Advantages and limits of application

The use of ultrathin cryosections for ultraimmunohistochemical investigations has gained in importance as technical and methodological improvements have been achieved. The well-known disadvantages of the synthetic resin technique (e.g. antigen denaturation, washing effects) have made it necessary to look for a more gentle method. In fact, material thus prepared offers the advantage that artefacts caused by dehydration and embedding in synthetic resin can be avoided. Besides, it also has a higher antigenicity compared with material prepared by means of the synthetic resin technique. Due to the different steps of incubation one has to face loss of substance and orientation when employing cryoprotected samples. Therefore it would be best to employ non-cryoprotected samples but this is nearly impossible in the case of ultraimmunohistochemical investigations because recrystallization of ice in the tissue leads to destruction of the ultrastructure. In order to quantify the extent of the changes we have compared the elemental distribution for several light elements of cryoprotected material, which was prepared following the Tokuyasu-technique, with non-cryoprotected samples. The latter were frozen in cryogen and the ultrathin sections were carefully freeze-dried. Although the loss caused by washing is low for membrane-linked structures, cryoprotected samples lose on average 50% of the analysed elements in the course of preparation in comparison with unprotected and only freeze-dried samples.