Journal of Nippon Medical School: Vol.71 No.5 page.306

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ArticleTitle Thin Is Better!: Ultrathin Cryosection Immunocytochemistry

AuthorList Toshihiro Takizawa¹ and John M. Robinson²

Affiliation ¹Department of Anatomy, Nippon Medical School
²Department of Physiology and Cell Biology, Ohio State University

Language EN

Volume 71

Issue 5

Year 2004

Page 306-307

Received

Accepted

Keywords

Abstract In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry^1-3. Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol¹. Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules^2,3.

Correspondence to Toshihiro Takizawa, MD, PhD, Department of Anatomy, Nippon Medical School, 1-1-5 Sendagi, Bunnkyo-ku, Tokyo 113-8602, Japan
t-takizawa@nms.ac.jp

ArticleTitle	Thin Is Better!: Ultrathin Cryosection Immunocytochemistry
AuthorList	Toshihiro Takizawa¹ and John M. Robinson²
Affiliation	¹Department of Anatomy, Nippon Medical School ²Department of Physiology and Cell Biology, Ohio State University
Language	EN
Volume	71
Issue	5
Year	2004
Page	306-307
Received
Accepted
Keywords
Abstract	In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry^1-3. Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol¹. Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules^2,3.
Correspondence to	Toshihiro Takizawa, MD, PhD, Department of Anatomy, Nippon Medical School, 1-1-5 Sendagi, Bunnkyo-ku, Tokyo 113-8602, Japan t-takizawa@nms.ac.jp