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Application of Cryotechniques with Freeze-substitution for the Immunohistochemical Demonstration of Intranuclear pCREB and Chromosome Territory

Nobuhiko Ohno, Nobuo Terada, Shin-ichi Murata, Ryohei Katoh and Shinichi Ohno

Departments of Anatomy (NO,NT,SO) and Pathology (SM,RK), Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi, Japan

Correspondence to: Shinichi Ohno, MD, PhD, Professor and Chairman, Dept. of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. E-mail: sohno{at}yamanashi.ac.jp

Intranuclear localization of signal molecules and chromosome territories has become more attractive in relation to postgenomic analyses of cellular functions. Cryotechniques and freeze-substitution (CrT-FS) have been generally used for electron microscopic observation to obtain better ultrastructure and immunoreactivity. To investigate benefits of applying the CrT-FS method to immunostaining of intranuclear signal molecules and FISH for chromosome territories, we performed an immunohistochemical study of phosphorylated cAMP-responsive element binding protein (pCREB) in mouse cerebellar tissues and a FISH study of chromosome 18 territory in human thyroid tissues using various cryotechniques. The immunoreactivity of pCREB was more clearly detected without antigen retrieval treatment on sections prepared by the CrT-FS method than those prepared by the conventional dehydration method. In the FISH study, more definite probe labeling of the chromosome territory could be obtained on paraffin sections by the CrT-FS method without microwave treatment, although such labeling was not clear even with microwave treatment on sections prepared by the routine dehydration method. The CrT-FS preserved relatively native morphology by preventing shrinkage of nuclei, and produced better immunoreactivity. Because the reduction of routine pretreatments in the present study might reveal more native morphology, the CrT-FS method would be a useful technique for intranuclear immunostaining and FISH. (J Histochem Cytochem 53:55–62, 2005)

Key Words: quick freezing • freeze-substitution • in vivo cryotechnique • antigen retrieval • pCREB • FISH • chromosome territory

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