Detection of injected fluorescence-conjugated IgG in living mouse organs using “in vivo cryotechnique” with freeze-substitution

Abstract

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 degrees C) cooled in liquid nitrogen over these living organs. The organs were subsequently freeze-substituted in acetone containing paraformaldehyde at about -80 degrees C, then gradually brought up to a room temperature, infiltrated with 30% sucrose and refrozen. Some well-frozen areas 300-400 mum 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.

Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

“…We have already reported novel Wndings about the dynamically changing structures of cells and tissues in living animal organs (Ohno et al 2004a). Recently, we also demonstrated that IVCT could be successfully applied to immunohistochemical studies, and was useful for analyzing easily translocated signal molecules (Zea-Aragon et al 2004;Terada et al 2004Terada et al , 2005Terada et al , 2006Li et al 2005;Ohno et al 2005a;Liao et al 2006). However, IVCT has not yet been applied to liver organs of living animals.…”

“…Thereafter, the frozen liver tissues were removed with a dental electric drill in liquid nitrogen. The frozen specimens for light microscopy were freezesubstituted with 2% PFA in acetone and embedded in either paraYn wax or OCT compound, as described before (Ohno et al 2004c;Terada et al 2005). The other specimens for electron microscopy were freeze-substituted in acetone containing 2% osmium tetroxide, and embedded in epoxy resin (Ohno et al 1996b).…”

Raman Microscopy of Freeze‐dried Mouse Eyeball‐slice in Conjunction with the “in vivo Cryotechnique”

“…It has also been difficult to examine the distribution of drugs in tissues after absorption or injection. We recently reported that the IVCT could be used to visualize injected fluorescence-conjugated target proteins (Terada et al, 2005). Raman spectroscopy has as well been applicable to detecting exogenous substances, such as caffeine, aspirin, barbital (Day et al, 2004), or ibuprofen (Breitenbach et al, 1999).…”

Immunohistochemical Application of Cryotechniques to Native Morphology of Cells and Tissues

“…5b, d, f). The concept that the in vivo cryotechnique retains soluble proteins in the animal tissues is applicable to other labeling techniques with fluorescent probes, and we have already demonstrated that they can retain their specific fluorescence character for the excitation and emission wavelengths even after quickfreezing followed by freeze-substitution and/or freezedrying [17]. Moreover, directly labeled proteins, such as green fluorescent protein (GFP), can also be used for the in vivo cryotechnique as a gene transfer animal model (manuscript in preparation).…”