大野 伸彦

Hereditary spastic paraplegia (HSP) is a neurological syndrome characterized by degeneration of central nervous system (CNS) axons. Mutated HSP proteins include myelin proteolipid protein (PLP) and axon-enriched proteins involved in mitochondrial function, smooth endoplasmic reticulum (SER) structure, and microtubule (MT) stability/function. We characterized axonal mitochondria, SER, and MTs in rodent optic nerves where PLP is replaced by the peripheral nerve myelin protein, P0 (P-0-CNS mice). Mitochondrial pathology and degeneration were prominent in juxtaparanodal axoplasm at 1 mo of age. In wild-type (WT) optic nerve axons, 25% of mitochondria-SER associations occurred on extensions of the mitochondrial outer membrane. Mitochondria-SER associations were reduced by 86% in 1-mo-old P-0-CNS juxtaparanodal axoplasm. 1-mo-old P-0-CNS optic nerves were more sensitive to oxygen-glucose deprivation and contained less adenosine triphosphate (ATP) than WT nerves. MT pathology and paranodal axonal ovoids were prominent at 6 mo. These data support juxtaparanodal mitochondrial degeneration, reduced mitochondria-SER associations, and reduced ATP production as causes of axonal ovoid formation and axonal degeneration.

Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1 day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1 day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine.

Advancement of microscopic technologies established significant progress in our understanding of the brain. In the recent effort to elucidate the complete wiring map of the brain circuitry termed 'connectome', the different modalities of imaging technology, including those of light and electron microscopy, have started providing essential contribution in multiple organisms. The contribution would be impossible without the recent innovation in both acquisition and analyses of the big connectomic data. The current data demonstrated complicated networks with unidirectional and reciprocal connections of the cerebral circuits at the macroscopic and light microscopic ('mesoscopic') levels, and the unimaginable complexity of synaptic connections between axons and dendrites at the electron microscopic ('microscopic') level. At the same time, the data highlighted the necessity to make substantial advancement in methodology of the connectomic studies, including efficient handling and automated analyses of the acquired dataset. Further understanding about structural and functional connectome seems to be facilitated by combinations of the different imaging modalities. Such multidisciplinary approaches will give us the clues to address whether the complete connectome can elucidate fundamental mechanisms processing the basic and higher functions of human brains.

Membrane skeletal networks form a two-dimensional lattice structure beneath erythrocyte membranes. 4.1R-MPP (membrane palmitoylated protein) 1-glycophorin C is one of the basic molecular complexes of the membrane skeleton. An analogous molecular complex, 4.1G-MPP6-cell adhesion molecule 4 (CADM4), is incorporated into the Schmidt-Lanterman incisure (SLI), a truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system. In this review, the dynamic structure of peripheral nerve fibers under stretching conditions is demonstrated using in vivo cryotechnique. The structures of nerve fibers had a beaded appearance, and the heights of SLI circular-truncated cones increased at the narrow sites of nerve fibers under the stretched condition. The height of SLI-truncated cones was lower in 4.1G-deficient nerve fibers than in wild-type nerve fibers. 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. The signal transduction protein, Src, was also involved in the 4.1G-MPP6-CADM4 molecular complex. The phosphorylation of Src was altered by the deletion of 4.1G. Thus, we herein demonstrate a membrane skeletal molecular complex in SLI that has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cells.

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.

During early development of the peripheral nervous system, Schwann cell precursors proliferate, migrate, and differentiate into premyelinating Schwann cells. After birth, Schwann cells envelop neuronal axons with myelin sheaths. Although some molecular mechanisms underlying myelination by Schwann cells have been identified, the whole picture remains unclear. Here we show that signaling through Tyro3 receptor tyrosine kinase and its binding partner, Fyn nonreceptor cytoplasmic tyrosine kinase, is involved in myelination by Schwann cells. Impaired formation of myelin segments is observed in Schwann cell neuronal cultures established from Tyro3-knockout mouse dorsal root ganglia (DRG). Indeed, Tyro3-knockout mice exhibit reduced myelin thickness. By affinity chromatography, Fyn was identified as the binding partner of the Tyro3 intracellular domain, and activity of Fyn is down-regulated in Tyro3-knockout mice, suggesting that Tyro3, acting through Fyn, regulates myelination. Ablating Fyn in mice results in reduced myelin thickness. Decreased myelin formation is observed in cultures established from Fyn-knockout mouse DRG. Furthermore, decreased kinase activity levels and altered expression of myelination-associated transcription factors are observed in these knockout mice. These results suggest the involvement of Tyro3 receptor and its binding partner Fyn in Schwann cell myelination. This constitutes a newly recognized receptor-linked signaling mechanism that can control Schwann cell myelination.

We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 mu m away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Ig.), J-chain and albumin. By cryotechniques, IgA immunoreactivitywas detected as tiny dotlike patterns in cytoplasm of some epithelial cells. Both J-chain and Ig. immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of livingmouse small intestines.

In postnatal development of the peripheral nervous system (PNS), Schwann cells differentiate to insulate neuronal axons with myelin sheaths, increasing the nerve conduction velocity. To produce the mature myelin sheath with its multiple layers, Schwann cells undergo dynamic morphological changes. While extracellular molecules such as growth factors and cell adhesion ligands are known to regulate the myelination process, the intracellular molecular mechanism underlying myelination remains unclear. In this study, we have produced Schwann cell-specific conditional knockout mice for cytohesin-2, a guanine-nucleotide exchange factor (GEF) specifically activating Arf6. Arf6, a member of the Ras-like protein family, participates in various cellular functions including cell morphological changes. Cytohesin-2 knockout mice exhibit decreased Arf6 activity and reduced myelin thickness in the sciatic nerves, with decreased expression levels of myelin protein zero (MPZ), the major myelin marker protein. These results are consistent with those of experiments in which Schwann cell-neuronal cultures were treated with pan-cytohesin inhibitor SecinH3. On the other hand, the numbers of Ki67-positive cells in knockout mice and controls are comparable, indicating that cytohesin-2 does not have a positive effect on cell numbers. Thus, signaling through cytohesin-2 is required for myelination by Schwann cells, and cytohesin-2 is added to the list of molecules known to underlie PNS myelination.

In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca2+ and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca2+ and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca2+ and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease. (c) 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 369-387, 2015

Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject.

The ultrastructures of the cerebellar cortex in living mice were investigated using the “in vivo cryotechnique” (IVCT). Electron microscopic observation with IVCT revealed that large extracellular spaces (ECS) were clearly observed among profiles of neurons and glia in molecular and Purkinje cell layers of the mouse cerebellum. The ECS were significantly reduced by prolonged ischemia/anoxia, since the ECS were smaller in tissue specimens obtained with IVCT following 8 min ischemia or the conventional quick-freezing method of resected fresh tissues. The parallel fibers observed with IVCT after short period of ischemia were slightly larger than those without ischemia, indicating cellular swelling after ischemia/anoxia. ECS were also abundant around synaptic terminals, which were totally or partially covered with glial processes, and the ECS sizes of opened (not covered with glial processes) and enclosed (covered with glial processes) synapses are estimated to be similar. These findings indicate that IVCT is useful to analyze ECS morphology in the central nervous system. The abundant ECS would allow morphological changes of neuronal and glial cells involved in dynamic remodeling and signal transduction of synapses.

It has been difficult to clarify the precise localizations of soluble serum proteins in thymic tissues of living animals with conventional immersion or perfusion fixation followed by alcohol dehydration owing to ischemia and anoxia. In this study, “in vivo cryotechnique” (IVCT) followed by freeze-substitution fixation was performed to examine the thymic structures of living mice and immunolocalizations of intrinsic or extrinsic serum proteins, which were albumin, immunoglobulin G1 (IgG1), IgA, and IgM, as well as intravenously injected bovine serum albumin (BSA). Mouse albumin was more clearly immunolocalized in blood vessels and interstitial matrices of the thymic cortex than in tissues prepared by the conventional methods. The immunoreactivities of albumin and IgG1 were stronger than those of IgA and IgM in the interstitium of subcapsular cortex. The injected BSA was timedependently immunolocalized in blood vessels and the interstitium of corticomedullary areas at 3.5 h after its injection and then gradually diffused into the interstitium of the whole cortex at 6 and 12 h. Thus, IVCT revealed defi nite immunolocalizations of serum albumin and IgG1 in the interstitium of thymus of living mice, indicating different accessibility of serum proteins from the corticomedullary areas, not from the subcapsular cortex of living animals, depending on various molecular sizes and concentrations.

In this chapter, we present time-dependent molecular states of rhodopsin (Rho) phosphorylation in mouse retina with “in vivo cryotechnique” (IVCT). Whole eyeballs of living mice under various dark- and light-exposure conditions were quickly frozen with isopentane–ropane cryogen cooled down in liquid nitrogen (−196 °C). The frozen whole-mount eyeballs were freeze-substituted and embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated 334Ser Rho (P-Rho334) antibody. Immunoreactivity of P-Rho334 was specifically obtained in the outer segments of mouse retinas exposed to daylight, and it was eliminated in the dark-adapted retinas. In the eyeballs exposed to strong visible light for 10 s after the dark adaptation, it was not detected. However, after 30, 60, and 180 s of visible light exposure, P-Rho334 immunoreactivity was recovered. Thus, IVCT revealed the time-dependent changes of the Rho phosphorylation in living mice within seconds following exposure to light.

A new “cryobiopsy” (CB) technique has been invented and applied for mice livers in vivo without stopping their blood circulation. They were routinely freeze-substituted in acetone containing fixatives for light microscopy (LM) or freeze-fractured before freeze substitution for scanning electron microscopy (SEM). Serial paraffin sections were stained with hematoxylin-eosin (HE) or histochemical periodic acid-Schiff (PAS) reaction. By HE-staining, the tissue surface areas were often compressed and sinusoidal erythrocytes got aggregated side by side. But in a little deeper tissue areas, hepatic sinusoids were widely open with flowing erythrocytes. Lots of PAS-reaction products were well preserved in hepatocytes of the CB specimens. To the contrary, they were unevenly distributed in hepatocytes of conventionally quick-frozen specimens or often lost in those of conventional dehydrated specimens. By SEM, some cell organelles, open Disse's spaces, and bile canaliculi appeared under normal blood circulation samples. The new CB technique would be useful for examining time-dependent morphological changes of functioning organs, including the livers, from an identical living animal.

In this chapter, we present application of “in vivo cryotechnique” (IVCT) to the injection of fluorescence-conjugated protein into living organs. The fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin antibody (FITC-IgG) was directly injected into mouse livers, which were then frozen with IVCT by pouring an isopentane-propane mixture (−193 °C) cooled in liquid nitrogen. The organs were subsequently freeze-substituted in acetone containing paraformaldehyde, infiltrated with sucrose, and cryocut into several slices. The slices were observed under the fluorescence microscope. By examining the distribution of FITC-IgG, aspects of functional blood circulation in the liver, such as the concept of the liver lobule, were confirmed. 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 same sections with different color images.

In vivo cryotechnique (IVCT) has been useful to examine native immunolocalizations of soluble proteins in animal kidneys, reflecting their living states. By using them, we have examined some immunolocalized proteins in nephrons of rat kidneys under experimental nephropathy induced by puromycin aminonucleoside (PAN) administration. At day 3 or day 9 after intraperitoneal PAN injection, IVCT was routinely performed for kidneys of anesthetized rats and followed by freeze-substitution fi xation. Serial tissue sections were first stained with hematoxylin-eosin (HE) and then immunostained for serum albumin and IgG1. The excretion of urinary proteins began to increase at day 3. Plasma protein analyses indicated the typical nephrosis. At day 3, although little morphological changes were detected by HE staining, both albumin and IgG1 were immunolocalized along cytoplasm of podocytes and in apical parts of epithelial cells of proximal convoluted tubules. At day 9, intravascular congestion of erythrocytes was observed in almost all of the glomerular basement membranes (GBMs). Their relative immunoreactivities of albumin and IgG1 were weaker within blood vessels including GBMs. IVCT revealed dynamically changing processes of protein excretion and reabsorption in various segments of nephrons.

Soluble proteins and glycogen particles are well preserved in paraffin-embedded sections prepared by in vivo cryotechnique (IVCT) followed by freeze-substitution (FS) fixation. We performed confocal laser scanning microscopic analyses on the distributions of glycogen with periodic acid–Schiff (PAS) staining and serum proteins with immunostaining for mouse liver tissues. Livers showed a red fluorescence signal of PAS staining in hepatocytes and immunofluorescence of immunoglobulin kappa (Igk) light chain in blood vessels and bile canaliculi. By three-dimensional (3D) reconstruction of serial optical sections, interconnecting hepatic sinusoids and bile canaliculi were detected with Igk immunostaining between trabecular hepatocytes that were PAS stained. In addition, 100-μm-thick PAS or eosin-stained slices provided 3D-structural images more than 30 μm in thickness away from tissue surfaces, showing blood vessels with flowing erythrocytes and networks of bile ducts and canaliculi. IVCT with histochemical analyses enabled us to visualize native hepatocytic glycogen and 3D structures, such as vascular networks, reflecting their living states by confocal laser scanning microscopy.

Soluble proteins and glycogen particles, which are easily lost upon conventional chemical fixation, have been reported to be better preserved in paraffin-embedded sections by “cryobiopsy” combined with freeze-substitution fixation (FS). In this part, we mentioned the distribution of glycogen in living mouse livers under (1) food restriction and (2) local circulation loss conditions with periodic acid-Schiff (PAS) staining by cryobiopsy. (1) Livers of fully fed mice showed high PAS-staining intensity in the cytoplasm of all hepatocytes, showing much glycogen in all hepatocytes. However, treatment with α-amylase clarifies that hepatocytes around the central vein preserve more glycogen than the hepatocytes around the portal vein. At 6 or 12 h after fasting, PAS-staining intensity markedly decreased in restricted areas of zone I near the portal tracts. This result was the same as that of specimens gotten from individual mouse. On the other hand, glycogen distributions were different in local circulation loss condition which caused by temporarily clipping of liver tissues followed by recovery of blood circulation. (2) In the liver tissues in which blood was recirculated for 1 h after the 30-min anoxia, PAS staining was still observed in zone II and also in restricted areas of zone I far from the portal tracts. We propose that the level of consumption or production of glycogen particles could vary in zone I, depending on the distance from the portal tracts. Thus, cryobiopsy combined with FS enabled us to examine time-dependent changes in glycogen distribution in the liver tissues of living mice. This combination might be applicable to the clinical evaluation of human liver tissues.

In this chapter, we present application of “in vivo cryotechnique” (IVCT) to measurement of oxygen saturation (SO2) in erythrocytes with Raman cryomicroscopy. Two resonance Raman (RR) shifts around 1355 and 1378 cm−1 which reflect deoxygenated and oxygenated hemoglobin molecular structures, respectively, were evaluated. The two RR shifts of quickly frozen human whole blood were well retained at the low temperature. In blood vessels of living mouse organs prepared with the IVCT, their RR spectral peaks were also detected at the same RR shifts. In the blood vessels of IVCT small intestines and livers, different peak patterns of their RR shifts were monitored in each blood vessel. Thus, SO2 in blood vessels is measurable in IVCT tissues with Raman cryomicroscopy, reflecting their living states.

In this chapter, we present application of “in vivo cryotechnique” (IVCT) to the injection of glutathione (GSH)-coated quantum dots (QDs), which emit a red-fluorescent signal with an ultraviolet excitation, for kidneys. The frozen kidneys were processed to freeze-substitution fixation (FS) and observed in fluorescence or confocal laser scanning microscope (CLSM). In the renal cortex, QD distribution was detected mostly in glomerular blood capillaries for a few seconds and extended to peritubular blood capillaries at 5 s. By injection of horseradish peroxidase (HRP) and QDs at the same time, the distribution of HRP in the renal cortex at 30 s was detected in the interstitium in addition to blood vessels, whereas QDs were localized only inside blood vessels. Thus, strict time-dependent visualization of blood flow in tissue sections became possible within seconds by combination of IVCT and injection of QDs.

Angiotensin II (AT) receptors, including AT receptor type 1 (AT1R) and type 2 (AT2R), are expressed in the rodent central nervous system, but their distributions and activation states are still unclear. In this study, we have performed immunohistochemical analyses of AT receptors in mouse cerebellum using our “in vivo cryotechnique” (IVCT). We used antibodies against amino-terminal domains of AT receptors, which are considered to undergo conformational changes upon the binding of AT. Immunoreactivity of AT1R was detected in mouse cerebellum and was highest in the outer tissue areas of molecular layers using IVCT. Surprisingly, the AT1R immunoreactivity in the cerebellar cortex was remarkably reduced following 5 and 10 min of hypoxia. The correlation of localization with GFAP and also hypoxia-induced decrease of its immunoreactivity were similarly observed by immunostaining of AT2R in the cerebellar specimens. These findings demonstrated that IVCT is useful to reveal dynamically changing immunoreactivities usually affected by receptor-ligand binding as well as hypoxia and also suggested that functional activities of AT receptors are time-dependently modulated under hypoxia in the central nervous system in comparison with the adrenal gland.

The wavelength of Raman-scattered light depends on the molecular composition of the substance. In this chapter, we present identification of biological molecules by Raman spectra in a mouse eyeball with “in vivo cryotechnique” (IVCT) followed by freeze drying. Four typical patterns of Raman spectra were obtained and electronically mapped. Tissue organization was confirmed by embedding the same eyeball slice used for Raman spectra into epoxy resin with the inverted capsule method. Each Raman spectral pattern represents a different histological layer in the eyeball. Some of the peaks of the Raman spectra obtained from the blood vessels in sclera and the photoreceptor layer were similar to those obtained from the purified hemoglobin and rhodopsin proteins, respectively. Thus, this method is useful for examining the distribution of a biological structures and chemical components.

Many molecular components and structures of living animal or human organs in biological and medical fi elds are dynamically changing for their necessary functions among lots of extracellular or intracellular body fluid. The importance of cryofi xation for morphology and immunohistochemistry, as already described in this book, is based on the way how to change the water contents into tiny vitreous ice crystal s. Our ultimate goal as morphological scientists is to obtain the real histology and pathology of living animal organ s including humans and also immunohistochemical localizations of functional molecules in their cells and tissues.

The transport mechanism of soluble molecules throughout the interstitial matrix is closely associated with human tumor behavior in vivo. However, the examination of soluble components in histological architectures has been hampered by artifacts caused during conventional tissue preparation. In this study, the immunodistribution of intrinsic and extrinsic serum components in tumor tissues was examined in xenografted human lung large carcinoma cells using “in vivo cryotechnique” (IVCT) and cryobiopsy, where target tissues are directly cryofixed in vivo. Human lung cancer cells were subcutaneously injected into the dorsal flank of nude mice, and paraffin sections and cryosections of produced tumors were prepared with different methods. Immunolocalization of serum proteins, including albumin, immunoglobulin G (IgG), and IgM, as well as intravenously injected bovine serum albumin (BSA) was examined. Their immunodistribution was more clearly observed in the interstitium by both IVCT and cryobiopsy than conventional methods. IgM was immunolocalized within blood vessels, whereas albumin and IgG were observed in the tumor interstitium. Moreover, intravenously injected bovine serum albumin exhibited leakage from the blood capillaries into surrounding connective tissues in 24 h, but it gradually diffused to the interstitium of the tumor masse in 3 days. These results suggest that molecular leakage from blood capillaries varies significantly in different areas of developing tumors and that small serum proteins, but not large ones, were abundantly immunolocalized in the tumor interstitium. Both IVCT and cryobiopsy were found to be useful for immunohistochemical studies of soluble molecules in tumors with blood circulation and may therefore be helpful for further histopathological analyses.

Protein kinases (PKs) phosphorylate proteins at active regions for signal transduction. In this study, normal and hypoxic mouse kidneys were prepared using an “in vivo cryotechnique” (IVCT) and examined immunohistochemically with specific antibodies against phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) to capture their time-dependent regulation in vivo. Left kidneys were cryofi xed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffi nized sections were immunostained for P-PK-S, Na + /HCO 3− cotransporter NBC1, and a membrane skeletal protein, 4.1B. The P-PK-S was diffusely immunolocalized in the cytoplasm of the proximal tubules in normal kidneys, whereas NBC1 and 4.1B were detected at the basal striations of S1 and S2 segments of the proximal tubule. After 10 or 30 s hypoxia, P-PK-S was still immunolocalized in the cytoplasm of kidneys, but it was detected at the basal striations after 1 or 2 min hypoxia. The immunolocalization of P-Akt-S was the same as P-PK-S in the normal and hypoxic kidneys. Immunoblotting analyses of the kidney tissues under normal or hypoxic condition clearly identifi ed the same 40 kDa bands. The IVCT is useful for timedependent analysis of the immunodistribution of P-PK-S and P-Akt-S.

In this chapter, we present the application of “in vivo cryotechnique” (IVCT) to examination of time-dependent topographical changes of leaking proteins from blood vessels in the mouse cerebellum, to assess the blood–brain barrier (BBB). The distribution of leaking serum proteins was compared by various cryotechniques. The cryofixed cerebellar tissues were processed for the freeze-substitution method and paraffin embedding. Serial deparaffinized sections were immunostained by anti-mouse immunoglobulin G (IgG) or albumin antibody. By combination of IVCT, serum IgG and albumin were clearly localized inside of cerebellar blood vessels. In anoxic cerebellar tissues, which were partially removed from brains in the mouse skull and quickly frozen in the isopentane–propane within a minute, serum IgG and albumin were diffusely immunostained around the blood capillaries, showing leakage of the serum components through the BBB changes. Thus, IVCT revealed in vivo localization of serum components in mouse brains.

Blood-follicle barrier (BFB) in ovarian follicles is the molecular sieve selective for size and charge. By utilizing the “in vivo cryotechnique” (IVCT), ovarian structures responsible for the BFB were analyzed during development of follicles under physiological conditions. Immunoreactivity of mouse serum proteins was better preserved with IVCT compared with other conventional methods. Strong immunoreactivity of albumin was detected in blood vessels, interstitium, and developing follicles. There was a clear alteration of the immunostaining intensity of IgG1 between inside and outside of the follicular basement membranes. Immunoreactivity of IgM was significantly changed between inside and outside of the vascular endothelial cells. These results suggest that permselectivity of BFB for soluble proteins with intermediate molecular weights is dependent on the follicular basement membrane and the vascular endothelial cells could play significant roles in the permselectivity for soluble proteins with high molecular weight.

Microscopic bioimaging of blood flow and distribution of cancer cells in lungs are essential to analyze mechanism of lung metastasis. Such cancer metastasis has been well known to induce hypercoagulable states and thrombosis. In histopathological tissue sections, however, it has been difficult to capture rapid phenomenon of thrombus formation due to technical problems associated with much less retention of soluble serum components as well as dynamic histological features reflecting their living states. In this part, to develop the cryotechnique for human pathology, we achieved bioimaging of both hypercoagulable states and thrombosis induced by early metastasis of mouse B16-BL6 melanoma. Thus, to examine plasma flow with fluorescence emission, glutathione-coated quantum dots (QDs) were subsequently injected after melanoma cells via right ventricles. At 5 s after the melanoma injection, melanoma cells were mostly stacked and intruded in alveolar capillaries with changing their shapes. Platelets aggregated around the stacked melanoma cells at 5 min. Such aggregated platelets were partially activated because of phosphorylated tyrosine 418 and 527 of Src on paraffin sections. Fibrin monomers and fibrinogens were also immunolocalized around stacked melanoma cells, indicating initial thrombus formation. In those areas, QDs were rarely detected, probably because of the lack of blood supply. Thus, IVCT revealed histopathological features of initial thrombosis under their circulatory conditions.

To elucidate alterations of follicular microenvironment and blood–follicle barriers (BFB) in polycystic ovary (PCO) syndrome, a mifepristone-induced PCO model was produced, and ovarian morphology and distribution of soluble plasma proteins were examined with in vivo cryotechnique (IVCT). Blood vessels with increased diameter and cystic follicles with degenerative membrana granulosa were clearly observed in the PCO model. The distribution of albumin (molecular weight, 69 kDa) and immunoglobulin M (IgM 900 kDa) was similar in normal and the PCO model ovaries. Albumin immunolocalized in the blood vessels, interstitium, and follicles, and IgM was mostly restricted within the blood vessels. In the PCO model, immunolocalization of immunoglobulin G (IgG 150 kDa), inter-alpha-trypsin inhibitor (ITI 220 kDa), and fibrinogen (340 kDa) was changed. In the PCO model ovaries, fibrinogen was mostly observed within blood vessels, and IgG and ITI were hardly observed in the membrana granulosa. The findings suggest that the PCO model ovaries are characterized by enhanced selectivity of the BFB in addition to increased blood flow, and some intermediate-sized molecules are blocked by follicular basement membranes. The pathogenesis and pathophysiology of PCO syndrome may be modulated by the hemodynamic conditions and permselectivity of BFB.

In living mouse kidneys, distributions of soluble proteins are usually difficult to analyzesing conventional tissue preparation methods. However, “in vivo cryotechnique“ (IVCT) followed by freeze-substitution is useful to examine immunolocalizations of the serum proteins in kidneys of bovine serum albumin (BSA)-overloaded mice. Following 2 days of daily intraperitoneal injection of BSA, proteinuria was evident, and BSA along with endogenous mouse albumin could be immunohistochemically detected in Bowman's space as well as urinary tubules of adult mouse kidneys with IVCT. Immunostaining for BSA and mouse albumin was detected in Bowman's space and proximal tubules of all nephrons, although IgG1 was not detected in about 45 % of nephrons. Immunolocalization of BSA and IgG1 observed with IVCT was different from those observed with the conventional preparation methods, which lack normal blood circulation of kidneys prior to fi xation. The immunolocalization of BSA and mouse serum proteins observed with IVCT suggests that glomerular filtration barriers in living mice are functionally impaired in a manner dependent on each nephron at the early stages of the BSA-overload mouse model.

To compare the influence of the different fixation procedures on the distribution of endogenous proteins of albumin and IgG in mouse glomeruli and identify the advantage of tissue fixation method in living mouse renal glomeruli by “in vivo cryotechnique” (IVCT), four different fixation methods were performed in mouse kidney tissues, such as (i) conventional immersion or (ii) quick-freezing following resected kidney tissues, (iii) quick-freezing following perfusion-fixation, and (iv) “in vivo cryotechnique” for living mouse kidneys. Kidne glomeruli were noticeably contracted after conventional immersion-fixation or quick-freezing following resection compared to glomeruli from tissues preserved by the IVCT. With the IVCT, both albumin and IgG were colocalized exclusively along or within the glomerular capillary loops however, immunoreactivity of these proteins in the other three methods was clearly detected in Bowman's space and apical cytoplasm of the proximal tubules. With the IVCT, immunoreactivity of collagen type IV was very weak at the glomerular basement membrane (GBM) until microwave treatment, which increased its immunoreactivity. Using conventional fixation method, aquaporin-1 (AQP-1) is abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs, but the proteins were prominent immunolocalization on the apical areas by “in vivo cryotechnique.” The “in vivo cryotechnique” should be a reliable tool to maintain soluble serum proteins and AQP-1 in situ and capture transient images of functioning kidney in living mice.

To overcome the problems of artifacts in the conventional immersion-fixation and dehydration method (IMDH), cryobiopsy and “in vivo cryotechnique” (IVCT), where target organs are directly cryofixed in vivo, were used for the analyses of microenvironment and histological architectures in xenografted human lung cancer tissues. The results revealed that tissue morphology, such as cellular organelles, and open blood vessels with flowing erythrocytes were clearly observed without artificial shrinkage using cryobiopsy and IVCT. By contrast, blood vessels were all collapsed following tissue resection in conventional methods. With cryobiopsy and IVCT, intravenously injected bovine serum albumin (BSA) clearly visualized functional blood vessels containing immunoglobulin M (IgM). The expression of vascular endothelial growth factor (VEGF) was correlated with the volume of blood vessels positive for IgM. The histological analyses of human tissues with cryobiopsy as well as IVCT will be useful to avoid artifacts inevitable in IMDH. Functional blood vessels along with relevant signal transduction would be precisely examined with cryobiopsy as well as IVCT to clarify microenvironment of the human tumors in clinical medicine.

In this chapter, we present application of in vivo cryotechnique (IVCT) samples to luciferin-luciferase reaction (LLR) for visualization of adenosine triphosphate (ATP), an energy source for muscle contraction. The LLR was clearly detected as an intentional design of the ATP attachment to glass slides with glutaraldehyde (GA). The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. In tissue sections of IVCT mouse skeletal muscles followed by freeze-substitution fixation (FS) in acetone containing GA, the LLR was detected in lumino-microsope. In muscle fibers and connective tissues, the intensity of LLR was different. Differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine reactions were detected in type I muscle fibers and erythrocytes in capillaries. Thus, it became possible to perform microscopic evaluation of the ATP distribution in the mouse skeletal muscles with IVCT.

Intestinal ischemia and ischemia/reperfusion rapidly progress to tissue destruction and reconstruction of functional organs. To date, precise immunolocalizations and the timing of appearance of cell-signaling components under such conditions have not been well visualized in conventional tissue sections. For immunohistochemistry, we have proposed that soluble molecules including cell-signaling proteins are well retained by our in vivo cryotechnique (IVCT). Mitogen-activated protein kinase (MAPK) signal transduction pathways have been reported to be activated under various types of cell damage, and cyclic AMP response element-binding protein (CREB) was directly phosphorylated with various cellular stimuli. In this study, both the expression and the immunolocalization of ERK1/2, a member of the MAPK family, were examined in mouse intestinal tissues by IVCT. Under normal conditions, although ERK1/2 was widely immunolocalized in the cytoplasm of epithelial cells, phosphorylated (p) ERK1/2 was slightly detected in a small amount of epithelial cells in crypt and top parts of the villi. In 20 min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. In 60 min ischemia, however, its immunoreactivity was remarkably increased in wide areas of epithelial cells. In these 20 min and 60 min ischemia groups, another phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. By ischemia 20 min reperfusion 60 min experiments, pERK1/2 immunointensity was reduced in the crypt areas. In 60 min ischemia with 60 min reperfusion, however, it was still strongly immunolocalized in epithelial cells of the crypts. Thus, rapidly changing ERK1/2 phosphorylation was visualized in the intestinal epithelial stem cells of mouse small intestine.

In vivo cryotechnique (IVCT) was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IMDH) and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG. Both albumin and BSA were well preserved in intercalated disks and T tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. IVCT has proved to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods.

Cryofixation and freeze-substitution (CF-FS) have been a technique to obtain better morphological data and immunoreactivity at the light and electron microscopic levels. Benefits of CF-FS have been demonstrated in immunostaining for an intranuclear antigen, phosphorylated cAMP-responsive element binding protein (pCREB), and fluorescence in situ hybridization (FISH), focusing on the chromosome territory. Compared with the conventional dehydration method, the higher immunoreactivity of pCREB was observed without the antigen retrieval treatment on sections prepared with the CF-FS methods. Probe labeling of No.18 chromosome territory in human thyroid tissues could be clearly observed on paraffin sections prepared with CF-FS without the microwave treatment, but not on sections prepared by the routine immersion fixation and dehydration method even with microwave treatment. The CF-FS method would be a useful technique for intranuclear immunostaining and FISH, since it preserves nuclear morphology by preventing shrinkage and achieves better signals.

The “in vivo cryotechnique” (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma.

Morphology and molecular distribution in animal liver tissues were examined using “in vivo cryotechnique” (IVCT) and compared with findings obtained with other conventional tissue preparation methods. Open sinusoids with flowing erythrocytes were clearly observed under normal blood circulation, and blood congestion and sinusoidal collapse were apparent in liver under ischemic or heart-arrest conditions. Sinusoidal collapse was also apparent with conventional tissue preparation methods, including immersion-fixation and quick-freezing (QF) methods of resected tissues, while perfusion-fixation artificially dilated the sinusoidal cavities. The IVCT and QF method well maintained immunoreactivity of soluble serum proteins including albumin and immunoglobulins as well as glycogen in hepatocytes detected by periodic acid-Schiff (PAS) staining. Rapid molecular translocation of serum proteins into hepatocytes was immunohistochemically observed after tissue resection with the QF method. IVCT was useful to examine dynamic morphology under different hemodynamic states and also immunodistribution of soluble components in living mouse livers under physiological and pathological states with higher time resolution.

In this chapter, we present application of “in vivo cryotechnique” (IVCT) to evaluation of blood-testis barrier (BTB) function in mouse testis, by visualization of albumin distribution. The albumin in the seminiferous tubules was well immobilized by combination of IVCT, freeze-substitution fixation and paraffin-embedding processes. In normal seminiferous tubules, albumin was immunostained as archlike pattern around some spermatogonia in basal compartments of seminiferous tubules, as well as in blood vessels and around peritubular myoid cells and Leydig cells. After the BTB disruption induced by injection of cadmium (Cd), some enlarged spaces and vesicular formations in the seminiferous epithelium were observed on the HE-stained sections. The albumin immunolocalization was detected not only in the basal compartments but also in the adluminal compartments between Sertoli cells and germ cells. Thus, the structural disruptions of BTB could be clearly demonstrated by IVCT.

Pimonidazole irreversibly binds to hypoxic cells with low oxygen tension in living animal organs. In this chapter, we present distribution of hypoxic cells in mouse liver tissues with “in vivo cryotechnique” (IVCT) by immunostaining for protein adducts of reductively activated pimonidazole (PARaPi). Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were immobilized by IVCT. The frozen liver tissues were freeze-substituted and immunostained for PARaPi. In liver tissues with 30 s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. Thus, IVCT revealed hypoxic cells with improved time resolution in tissue sections.

Tumor behavior depends on the complex tumor interstitium and microenvironment, which influence transport of fluid and soluble molecules from blood vessels. The purpose of this study was to reveal how complex tumor tissues affect the immunodistribution of serum proteins and time-dependent translocation of bovine serum albumin (BSA) from blood vessels, using relatively differentiated human adenocarcinoma produced by the xenografted A549 cell line. Histological architecture and immunodistribution of the serum proteins in adenocarcinomatous tissues were clearly detected by the in vivo cryotechnique and cryobiopsy. Both albumin and IgG1 were detected in blood vessels, connective tissues around the tumor mass, and the interstitium among tumor cell nests. IgM was mainly detected in blood vessels and connective tissues around the tumor mass but was not detected in the interstitium among the tumor cell nests. At 10 or 30 min after BSA injection, BSA was observed only in blood vessels, but 1 h after the injection, it was also detected in the interstitium and surrounding connective tissues of the tumor mass. The present findings showed topographic variation of molecular permeation in the adenocarcinomatous tumor mass. The interstitial tissues with augmented permeability of serum proteins would increase accessibility of tumor cells to blood-derived molecules.

The “in vivo cryotechnique“ (IVCT) is useful to capture dynamic blood flow conditions. We injected various concentrations of horseradish peroxidase (HRP) into anesthetized mice via left ventricles to examine architectures of thymic blood vessels and their permeability at different time intervals. At 30 s after HRP injection, enzyme reaction products were weakly detected in interstitium around some thick blood vessels of corticomedullary boundary areas, but within capillaries of cortical areas. At 1 and 3 min, they were more widely detected in interstitium around all thick blood vessels of the boundary areas. At 10 min, they were diffusely detected throughout interstitium of cortical areas and more densely seen in medullary areas. At 15 min, however, they were uniformly detected throughout interstitium outside the blood vessels. At 30 min, phagocytosis of HRP by macrophages was scattered throughout the interstitium, which was accompanied by the decrease of HRP reaction intensity in interstitial matrices. Thus, time-dependent HRP distributions in living mice indicate that molecular permeability and diffusion depend on different areas of thymic tissues.

We performed immunohistochemical or ultrastructural analyses of living mouse small intestines prepared by “in vivo cryotechnique” (IVCT). Living morphological states of small intestinal tissues, including flowing erythrocytes and opening blood vessels, were observed on paraffin-embedded sections prepared with IVCT. IgA was immunolocalized in many plasma cells of the lamina propria mucosa, intestinal matrices, and also in epithelial cells of the intestinal villi and crypts. Both IgG1 and IgM immunoreactivities were mainly detected in blood vessels, whereas only IgG1 was also immunolocalized in interstitial matrices of mucous membranes. Confocal laser scanning micrographs of doublefluorescence immunostaining for IgA immunoreactivity are detected in the cytoplasm of epithelial cells as well as plasma cells in the lamina propria mucosa. On the other hand, by electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5–10 μm away from the cryogen-contact surface tissues. Apical microvilli of epithelial cells contained dynamically waving actin filaments. Furthermore, highly electrondense organelles, such as mitochondria, in addition to endoplasmic reticulum and ribosomes, were well preserved under the widely organized terminal web. Additionally, Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval, and immunostained for various proteins, such as IgA, Igκ, IgG1, IgM, J-chain, and albumin. IgA immunoreactivity was detected as a tiny dot-like pattern in the cytoplasm of some epithelial cells and plasma cells localized in the lamina propria. The J-chain and Igκ immunoreactivities were also detected in the same local areas as those of IgA. Thus, IVCT was useful for the preservation of soluble serum proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.

Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. In this chapter, we present the contribution of Dyn2 to the structural integrity of discoid vesicles (DVs) and to the endocytic activity of urinary bladder umbrella cells (UCs) which have capacity to control bladder volume. Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Treatment of bladders with a Dyn-GTPase inhibitor, dynasore, markedly reduced the number of DVs in UCs. In infected UCs, Escherichia coli was encased in compartments enriched in Dyn2, and treatment of bladders with dynasore inhibits E. coli invasion of UCs.

The purpose of this study is to visualize topographical changes of serum proteins, albumin and IgG, passing through the mouse glomerular capillary loops and their reabsorption in renal proximal tubules by immunohistochemistry in combination with our “in vivo cryotechnique.” Under the acute hypertensive condition, the albumin and kappa or lambda light chains, but not IgG1 heavy chain, were strongly immunolocalized in the apical cytoplasm of almost all proximal tubules. This study is the first in vivo visualization for glomerular passage of serum proteins and their transtubular absorption.

To identify immunoglobulin (Ig)-producing cells with immunohistochemistry, conventional methods of preparation using chemical fixatives have problems such as the artificial diffusion of components and antigen masking. The “diffusion artifact” is caused by the translocation of soluble proteins like Ig from the serum to cytoplasm or vice versa. We have examined the immunolocalization of serum proteins, such as Ig kappa light chain (Igκ), IgG1 heavy chain (IgG1), and albumin, in immunized mouse spleens after a peritoneal injection of human hemoglobin. Better preservation of morphology and immunoreactivity was obtained with the “in vivo cryotechnique” (IVCT) followed by freeze-substitution than with conventional preparative methods. Although Ig-producing cells were not clearly detected in red pulp of 2-day-immunized spleens with the conventional methods, Igκ- immunopositive cells with rich cytoplasm were detected in the red pulp with IVCT, especially in the subcapsular and peritrabecular areas, where IgG1-immunopositive cells were rarely observed. In 7-day-immunized spleens prepared with IVCT, Igκ- or IgG1-immunopositive cells were mostly located in peritrabeculae. The development of Ig-producing cells was clarified in the specimens prepared with IVCT, which proved to be useful for analyzing the native morphology and distribution of Ig-producing cells.

In vivo cryotechnique (IVCT) followed by freeze substitution (FS) enabled us to analyze the cells and tissues reflecting living states. In this chapter, we present immunolocalization of glutamate (Glu) in the inner segment (IS) of photoreceptors in the mouse retina with IVCT, which was controversial with conventional fixation methods. Eyeballs from anesthetized mice were directly frozen using IVCT and processed for FS fixation in acetone containing chemical fixatives and embedded in paraffin. Deparaffinized sections were immunostained with an anti-Glu antibody. Strong Glu immunoreactivity was obtained in the specimens prepared by FS with a low concentration of glutaraldehyde, whereas no Glu immunoreactivity was obtained without the chemical fixatives. Thus, it became clear that Glu was immunolocalized in the IS, probably reflecting living states.

When tissue specimens are prepared for microscopic observation, commonly used methods using chemical fixatives and dehydration can yield artifacts in morphology and molecular distribution. Conventional quick-freezing and high-pressure freezing methods, where specimens are cryofixed after resection, can also cause artifacts due to anoxia and ischemia, and only dead morphological states without blood circulation can be observed with these methods. By contrast, all cells and tissues in animal bodies can be cryofixed in situ by “in vivo cryotechnique” (IVCT), which prevents these artifacts caused following removal of target tissues. In the central nervous system, distribution of small molecules, dynamic changes of signaling proteins, ultrastructures of extracellular space, and blood–brain barrier integrity have been examined using IVCT. To acquire novel information in cryofixed tissues, new imaging methods including Raman microscopy were combined with IVCT. New approaches to observe “living” states in the nervous system will be possible with IVCT.