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Journal of Electron Microscopy Advance Access published online on May 2, 2006
Journal of Electron Microscopy, doi:10.1093/jmicro/dfl011
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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 February 7, 2006
Accepted April 5, 2006

Full-length paper

Ultrahigh-resolution immunofluorescence microscopy using ultrathin cryosections: subcellular distribution of caveolin-1{alpha} and CD31 in human placental endothelial cells

Miki Mori 1, Gen Ishikawa 2, Toshiyuki Takeshita 2, Tadashi Goto 1, John M. Robinson 3, and Toshihiro Takizawa 1 *

1 Department of Molecular Anatomy, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan
2 Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo 113-8602, Japan
3 Department of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210, USA

* To whom correspondence should be addressed.
Toshihiro Takizawa, E-mail: t-takizawa{at}nms.ac.jp


  Abstract

We compared the z-axis resolutions achieved by immunofluorescence (IF) microscopic imaging of tissue sections of different thicknesses (ultrathin cryosections, optical sections of cryostat sections and conventional cryostat sections). We used these images to determine the distribution of caveolin-1{alpha} (CAV-1{alpha}) and CD31 in endothelial cells of full-term, human placenta. Anti-CAV-1{alpha} antibody was used to visualize caveolae, which are among the smallest organelles. By using ultrathin cryosections as substrates for IF microscopy, we were able to resolve discrete caveolae that were primarily present immediately beneath the endothelial cell surface. In contrast, neither conventional nor confocal images from cryostat sections were able to resolve individual caveolae, despite dramatic reductions in the confocal image degradation that arises from out-of-focus fluorescence signals. Anti-CD31 antibody labeled the endothelial cell surface exclusively. Quantitative analysis of ultrathin cryosections showed that about 2.5 times more CD31 was expressed on the luminal surface of cells than on the abluminal surface. Our results demonstrate that ultrathin cryosections can serve as excellent substrates for ultrahigh-resolution IF microscopy.

Keywords: Caveolin; CD31; fluorescence microscopy; human placenta; immunocytochemistry; ultrathin cryosections.
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