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Journal of Electron Microscopy Advance Access published online on June 16, 2006
Journal of Electron Microscopy, doi:10.1093/jmicro/dfl018
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© The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received March 3, 2006
Accepted May 22, 2006

Full-length paper

Morphological and histochemical analyses of living mouse livers by new ‘cryobiopsy’ technique

Yasuhisa Fujii 1, Nobuhiko Ohno 1, Zilong Li 1, Nobuo Terada 1, Takeshi Baba 1, and Shinichi Ohno 1 *

1 Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-shi, Yamanashi 409-3898, Japan

* To whom correspondence should be addressed.
Shinichi Ohno, E-mail: sohno{at}yamanashi.ac.jp


  Abstract

A new ‘cryobiopsy’ (CB) technique has been invented for freezing the functioning livers of living mice in vivo without stopping their blood circulation. Livers of anesthetized mice were pinched off with pre-cooled CB forceps and immediately plunged into isopentane-propane cryogen. They were routinely freeze-substituted in acetone containing paraformaldehyde for light microscopy (LM) or osmium tetroxide for scanning electron microscopy (SEM). By freeze-fracturing some of them with a scalpel in liquid nitrogen before the freeze-substitution, well-preserved tissue areas were exposed only for SEM. They were either embedded in paraffin wax for LM or infiltrated with t-butyl alcohol followed by freeze-drying for SEM. Serial paraffin sections were stained with hematoxylin-eosin (HE) or histochemical periodic acid-Schiff (PAS) reaction. By HE-staining, the tissue surface areas were often compressed with the CB forceps and sinusoidal erythrocytes became aggregated side by side. In slightly deeper tissue areas, however, hepatic sinusoids were widely open with flowing erythrocytes. Lots of PAS-reaction products were well preserved in hepatocytes of the CB specimens. On the contrary, they were unevenly distributed in hepatocytes of conventionally quick-frozen specimens, and often lost in those of the conventionally dehydrated specimens. By SEM, some cell organelles, such as mitochondria and endoplasmic reticulum, and also dilated fenestrae of endothelial cells, open Disse's spaces and bile canaliculi appeared to be under normal blood circulation in the prepared CB samples. The new CB technique would be easy and useful for repeated examination of functioning organs of a living animal.

Keywords: cryobiopsy; freeze-substitution; quick-freezing; PAS-reaction; scanning electron microscopy.
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