Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique

Ohno, Shinichi; Terada, Nobuo; Fujii, Yasuhisa; Ueda, Hideho; Takayama, Ichiro

doi:10.1007/bf00199513

Cited by 93 publications

(86 citation statements)

References 28 publications

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“…In the seminiferous tubules, as 4.1G was relatively immunolocalized as round and/or arch-shaped patterns in the basal compartment as well as honeycomb patterns in the adluminal compartment . In this study, we further evaluated the 4.1G immunolocalization at various stages of the mouse seminiferous cycle with 'in vivo cryotechnique (IVCT)' (Ohno et al 1996) and found different 4.1G distributions among the various stages. In addition, with immunoelectron microscopy, 4.1G was found to be immunolocalized not only in Sertoli cells, but also in a subset of germ cells including spermatogonium.…”

Section: Introductionmentioning

confidence: 99%

Involvement of a membrane skeletal protein, 4.1G, for Sertoli/germ cell interaction

Terada¹,

Ohno²,

Saitoh³

et al. 2010

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We previously reported that a membrane skeletal protein, 4.1G (also known as EPB41L2), is immunolocalized in mouse seminiferous tubules. In this study, the 4.1G immunolocalizaiton was precisely evaluated at various stages of the mouse seminiferous epithelial cycle with 'in vivo cryotechnique' and also with pre-embedding immunoelectron microscopy in testicular tissues whose ultrastructures were well preserved with glycerol treatment before cryosectioning. In addition, 4.1G-deficient mice were produced, and the morphology of their seminiferous tubules was also evaluated. The 4.1G immunolocalization was different among stages, indicating that it was not only along cell membranes of Sertoli cells, but also those of spermatogonia and early spermatocytes. To confirm the 4.1G immunolocalization in germ cells, in vitro culture of spermatogonial stem cells (SSCs) was used for immunocytochemistry and immunoblotting analysis. In the cultured SSCs, 4.1G was clearly expressed and immunolocalized along cell membranes, especially at mutual attaching regions. In testicular tissues, cell adhesion molecule-1 (CADM1), an intramembranous adhesion molecule, was colocalized on basal parts of the seminiferous tubules and immunoprecipitated with 4.1G in the tissue lysate. Interestingly, in the 4.1G-deficient mice, histological manifestation of the seminiferous tubules was not different from that in wild-type mice, and the CADM1 was also immunolocalized in the same pattern as that in the wild-type. Moreover, the 4.1G-deficient male mice were fertile. These results were probably due to functional redundancy of unknown membrane skeletal molecules in germ cells. Thus, a novel membrane skeletal protein, 4.1G, was found in germ cells, and considering its interaction with CADM family, it probably has roles in attachment of both Sertoli-germ and germ-germ cells.

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Section: Introductionmentioning

confidence: 99%

Involvement of a membrane skeletal protein, 4.1G, for Sertoli/germ cell interaction

Terada¹,

Ohno²,

Saitoh³

et al. 2010

Reproduction

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“…In vivo cryotechnique is a new and original method which can prevent morphological and immunohistochemical alterations caused by stopping of blood circulation that induces anoxia and rapid loss of blood volume and pressure [17,18,36]. Such a method can, therefore, be expected to enable more detailed immunohistochemical analyses of transient and dynamic morphology in living animal organs.…”

Section: Discussionmentioning

confidence: 99%

“…The lack of blood supply into some organs can easily modify their ultrastructure and molecular distributions, presumably induced by anoxia and rapid loss of blood volume and pressure [17,18,26,27,[30][31][32]35]. In addition, such loss of blood supply, inevitable in experimental studies involving conventional chemical fixation or common QF of fresh tissue specimens, sometimes modifies the immunoreactivity of various functional molecules, due to the rapid ischemia, which induces diverse responses in living animal organs [8,29].…”

Section: Significance Of Cryotechniques For Immunohistochemistrymentioning

confidence: 99%

“…Because in vivo cryotechnique can immediately cryofix cells and tissues of living animals, it avoids problematic changes due to stopping of blood circulation and resection of organs, and is useful for demonstrating not only ultrastructures of living animals with blood circulation but also quick and transient morphological changes in vivo without such unwanted effects [17,18,26,27,31]. Tissue samples directly frozen by in vivo cryotechnique can be processed in the same ways as those by other conventional QF methods, including slamming QF, plunging QF and high-pressure freezing, not only for electron microscopy, but also for light microscopy ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

Ohno

Terada

Ohno

2004

Acta Histochem. Cytochem.

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The quick-freezing method often used for physical fixation also contributed enormously to the advancement of morphology, but it could not provide enough information about the dynamic morphological changes in vivo in living animal organs. Therefore, we developed the in vivo cryotechnique in 1995, which could directly cryofix organs in vivo under anesthetized conditions without stopping blood circulation or producing effects of anoxia. We have already reported new findings about the in vivo ultrastructures of living animal organs with the cryotechnique followed by freeze-substitution or replica preparation. Recently, it has also been applied to other morphological analyses in vivo, including immunohistochemistry and FISH, for obtaining dynamically functioning structures of cells and tissues at a light microscopic level. In such experimental processes, the in vivo cryotechnique has been shown to have the added benefit of direct antigen-retrieval effects since it reduces several steps of retrieval treatments which are required in common paraffin-embedded samples prepared by the conventional fixation and dehydration. In conclusion, the in vivo cryotechnique allows us to investigate the functioning real morphology of living animals, which was technically difficult to be observed before, and perform dynamic immunohistochemistry of cells and tissues in various animal organs in vivo at ultimate time-resolution.

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“…Moreover, in the past decade, the in vivo cryotechnique has been developed to achieve better detection of in vivo morphology [4], using a combination of cooled metal cryoknife with isopentane-propane liquid cryogen. After in vivo freezing, followed by freeze-substitution or freeze-fracture replica method, the frozen specimens can be processed for light or electron microscopic observation, as shown in Figure 2.…”

Section: Introductionmentioning

confidence: 99%

Immunohistochemical Application of Cryotechniques to Native Morphology of Cells and Tissues

Terada

Ohno

2004

Acta Histochem. Cytochem

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Common immunohistochemistry is a technique based on antigen-antibody reactions in thin sections. It is well known that the important steps of tissue preparation protocols are (i) fixation (chemical and physical methods, especially cryofixation), (ii) sectioning, and (iii) immunostaining. We have already examined how to combine these preparation steps for an improved immunohistochemistry of target cells and tissues so as to overcome some of the technical problems commonly encountered. In many experimental cases, various cryotechniques were useful to obtain both a strong immunoreactivity of the antigens and a high time-resolution of the morphology of living animal cells and tissues. When the ultimate aim is to obtain in vivo observation of the morphology and immunolocalization, the new concept of in vivo cryotechnique becomes indispensable in the field of cell biology.

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Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique

Cited by 93 publications

References 28 publications

Involvement of a membrane skeletal protein, 4.1G, for Sertoli/germ cell interaction

Involvement of a membrane skeletal protein, 4.1G, for Sertoli/germ cell interaction

Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

Immunohistochemical Application of Cryotechniques to Native Morphology of Cells and Tissues

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