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

Abstract

Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using "in vivo cryotechnique" (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid-Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5 min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.

Histological Study and LYVE-1 Immunolocalization of Mouse Mesenteric Lymph Nodes with "In Vivo Cryotechnique"

“…Isopentane-propane cryogen (−193°C) cooled in liquid nitrogen was directly poured over mesenteric lymph nodes (Fig. 1b) while their hearts were beating, in a similar way to that for living mouse livers [21]. Then, the frozen lymph nodes were removed with a dental electric drill in liquid nitrogen (Fig.…”

“…IVCT can prevent morphological artifacts of cells and tissues caused by tissue-resection and immersion- or perfusion-fixation [1, 21, 31]. It is also possible to examine immuolocalization of soluble proteins, as well as small amino acids, with high immunoreactivity, reflecting their original localization [15, 21, 34, 39, 48]. In this study, we performed morphofunctional analyses of mouse mesenteric lymph nodes under normal blood circulation prepared with IVCT, and examined immunolocalization in situ of LYVE-1, type IV collagen and smooth muscle actin in the lymph nodes.…”

Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

“…The subsequent FS is thought to substantially preserve their morphology and localization with Wxatives at low temperatures by substituting hyaline ice crystals with organic solvents, such as acetone and alcohol, in which biological components are almost immobile during such a preparation step. In our previous studies, soluble proteins in blood vessels were demonstrated to be well preserved at their original sites under diVerent hemodynamic conditions Ohno et al 2006;Terada et al 2006b). For such immunohistochemical studies, tiny ice crystal formation during the freezing step also helped to increase the penetration of antibodies into deep tissues away from the tissue surface without special antigen-retrieval steps, such as the commonly used microwave treatment .…”

Application of “in vivo cryotechnique” to detect erythrocyte oxygen saturation in frozen mouse tissues with confocal Raman cryomicroscopy

“…For the past few years, it has provided not only morphology reflecting the living states of animal organs , but also structural molecular changes with higher time-resolution compared to those revealed by various conventional fixation methods (Terada et al, 2006c). In addition, soluble serum proteins localized in cells and tissues of living mouse organs were visualized with IVCT followed by a freeze-substitution fixation (FS) method for immunohistochemistry (Li et al, 2005(Li et al, , 2006Ohno et al, 2006;Zhou et al, 2007;Saitoh et al, 2008). However, some frozen biological components can move from their original sites in the cells and tissues of living animals and also change their molecular structures during the subsequent FS preparation after IVCT (Terada et al, 2006a).…”