Abstract
Cryofixation and freeze-substitution (CF-FS) have been a technique to obtain better morphological data and immunoreactivity at the light and electron microscopic levels. Benefits of CF-FS have been demonstrated in immunostaining for an intranuclear antigen, phosphorylated cAMP-responsive element binding protein (pCREB), and fluorescence in situ hybridization (FISH), focusing on the chromosome territory. Compared with the conventional dehydration method, the higher immunoreactivity of pCREB was observed without the antigen retrieval treatment on sections prepared with the CF-FS methods. Probe labeling of No.18 chromosome territory in human thyroid tissues could be clearly observed on paraffin sections prepared with CF-FS without the microwave treatment, but not on sections prepared by the routine immersion fixation and dehydration method even with microwave treatment. The CF-FS method would be a useful technique for intranuclear immunostaining and FISH, since it preserves nuclear morphology by preventing shrinkage and achieves better signals.
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Ohno, N., Terada, N., Ohno, S. (2016). Cryotechniques and Freeze-Substitution for Immunostaining of Intranuclear Antigens and Fluorescence In Situ Hybridization. In: Ohno, S., Ohno, N., Terada, N. (eds) In Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs. Springer, Tokyo. https://doi.org/10.1007/978-4-431-55723-4_39
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DOI: https://doi.org/10.1007/978-4-431-55723-4_39
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