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Detection of injected fluorescence‐conjugated IgG in living mouse organs using “in vivo cryotechnique” with freeze‐substitution
Author(s) -
Terada Nobuo,
Ohno Nobuhiko,
Li Zilong,
Fujii Yasuhisa,
Baba Takeshi,
Ohno Shinichi
Publication year - 2005
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20155
Subject(s) - in vivo , fluorescence , conjugated system , substitution (logic) , chemistry , microbiology and biotechnology , biology , biophysics , quantum mechanics , computer science , programming language , polymer , physics , organic chemistry
In this experiment, we performed the “in vivo cryotechnique” in tandem with fluorescence microscopy. The fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit immunoglobulin (IgG) antibody (FITC‐IgG) was directly injected into mouse livers or kidneys, which were then frozen in vivo by pouring an isopentane‐propane mixture (−193°C) cooled in liquid nitrogen over these living organs. The organs were subsequently freeze‐substituted in acetone containing paraformaldehyde at about −80°C, then gradually brought up to a room temperature, infiltrated with 30% sucrose and refrozen. Some well‐frozen areas 300–400 μm below the frozen tissue surface were cryocut into several slices. The slices were observed under the fluorescence microscope. By examining the distribution of FITC‐IgG in the frozen livers, some aspects of functional blood circulation in the liver, such as the concept of the liver lobule, were reconfirmed. This also confirmed that the blood flow in the liver after the FITC‐IgG injection was normal. The subsequent preparation of the specimens with immunohistochemistry, using the tetramethylrhodamine (TRITC)‐conjugated anti‐mouse IgG antibody, allowed us to visualize the localizations of both the original mouse IgG and the injected goat IgG in the cryosections with different color images. The experimental protocol presented demonstrates the in situ localization of the various proteins labeled with fluorescent probes, and it can, in conjunction with immunohistochemistry, localize proteins in cells and tissues. Microsc. Res. Tech. 66:173–178, 2005. © 2005 Wiley‐Liss, Inc.