Application of rapid-frozen substitution fixation method to immunohistochemistry

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A rapid-freezing substitution technique is a quite useful tool to study the ultrastructural features of cells in a more life-like state. However, there remained several difficulties in applying the technique to immunoelectron microscopy. In this study, we tried to overcome those difficulties to perform immunolabelling in well- preserved ultrastructure.Frozen rat parotid gland and exocrine pancreas tissue blocks were quenched by smaching against a copper block surface which was pre-cooled with liquid helium. The tissue blocks were freeze-substituted in either of the following media; osmium in acetone, paraformaldelhyde in acetone, and glutaraldehyde in acetone. Paraformaldehyde in acetone and glutaraldehyde in acetone were prepared with our new distillation device. After substitution, trimmed materials were embedded. Ultrathin sections were immunostained with rabbit anti-rat-amylase antibodies and protein A/colloidal gold.High labelling density was obtained from the materials which were fixed by freeze- substitution with glutaraldehyde in acetone or paraformaldehyde in acetone, embedded in Araldite 6005. In materials substituted with Os04 in acetone, embedded in an epoxy resin mixture and etched with sodium metaperiodate solution, labelling density was lowr than the former. In Lowicryl K4M-embedded materials labelling was the ghighest among the mataerials examined so far.The results in this study present an useful and preactical information in the application of immunocytochemistry to rapid-frozen substitution fixed tissues.