Quick-freezing And Deep-etching Method Research Articles

Extraskeletal myxoid chondrosarcoma: a study using a quick-freezing and deep-etching method

A case of extraskeletal myxoid chondrosarcoma (ESMC), which developed in the right thigh of a middle-aged Japanese woman, was studied using immunohistochemistry, conventional electron microscopy, and the quick-freezing and deep-etching (QF-DE) method. In addition to typical light microscopic findings of ESMC, conventional electron microscopy indicated that the tumor cells had features of chondrocytes. Immunohistochemically, the tumor cells showed a positive immunoreaction for S100 protein. A diagnosis of ESMC was made. An interesting observation was the ultrastructural features of collagen fibrils in the myxoid matrix highlighted by the QF-DE method. These collagen fibrils consisted of relatively thin collagen (20-35 nm) with pleated surface structures. The surface striation at 65 nm was obscure. We consider that such a finding of collagen fibrils identified by the QF-DE method is one of the characteristics of the myxoid matrix of ESMC, and this is useful for the differential diagnosis of myxoid soft tissue tumors.

Ultrastructural study of human glomerular capillary loops with IgA nephropathy using quick-freezing and deep-etching method.

Immunoglobulin A (IgA) nephropathy shows great variability regarding the histological features of the lesions of human renal glomeruli. In the present study, the quick-freezing and deep-etching (QF-DE) method was used to analyze the glomerular ultrastructure of biopsied kidney tissues from children with IgA nephropathy. Biopsied renal tissues were routinely prepared for light microscopy, immunofluorescence microscopy, conventional electron microscopy, and replica electron microscopy. The three-dimensional ultrastructure of glomeruli of the kidney was clearly observed by using the QF-DE method. Three layers of glomerular basement membranes, i.e., middle, inner and outer layers, were clearly detected in the replica electron micrographs. The middle layer was 343.0+/-24.2 nm (n=20) in width and formed polygonal meshwork structures. We also observed slit diaphragms, electron-dense mesangial deposits, and increased amounts of mesangial matrix and foot process effacement. Many delicate filaments were found to be distributed from the apical to the bottom portions between neighboring foot processes. The ultrastructural difference between the replica electron micrographs and conventional electron micrographs was found to be especially marked in the appearance of foot processes and connecting filaments between the neighboring foot processes. The examination of extracellular matrix changes, as revealed at high resolution by the QF-DE method, gave us some morphofunctional information relevant to the mechanism of proteinuria with IgA nephropathy.

Clinical applications of the quick-freezing and deep-etching method to human diabetic nephropathy

Mesangial expansion and glomerular basement membrane (GBM) thickening were not different between normoalbuminuric (NA) and microalbuminuric (MA) type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. In the present study, the QF-DE method was applied to the renal biopsy from 6 type 2 diabetic patients without definable renal diseases other than diabetic nephropathy. Four patients were NA and the other two were MA. Three control specimens were normal parts in surgically resected kidneys of renal cell carcinoma. Replica membranes were prepared by the QF-DE method as previously described. By the QF-DE method, both GBM middle layer and mesangial matrix (MM) were composed of polygonal meshwork structures. The mesh pores of GBM and MM were more enlarged in size and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The diameters of mesh pores in the diabetic patients were significantly larger than those in the control subjects. In conclusion, the QF-DE method could be applied to needle renal biopsy and the present study has firstly clarified the difference of ultrastructural changes between NA and MA type 2 diabetic patients, which had not been disclosed by the conventional electron microscopy, were revealed by the QF-DE method.

Application of a quick-freezing and deep-etching method to pathological diagnosis: a case of elastofibroma

A case of elastofibroma in a middle-aged Japanese woman was examined by the quick-freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient.

Histochemical study of apoptotic epithelial cells depending on testosterone in primary cultured rat prostatic tissues.

To clarify whether apoptosis can be induced in cultured rat prostatic epithelial cells, they were investigated at various time points, depending on different concentrations of testosterone. Ventral lobes of rat prostates were cultured as small pieces of tissues up to 14 days. They were examined by anti-Fas antibody immunostaining and also compared to findings revealed by in situ end-labelling (ISEL) technique. To clarify apoptotic nuclei at high resolution, the quick-freezing and deep-etching (QF-DE) method was also used, as reported before. The localization and appearance of Fas-positive cells were detected more widely and earlier than those of ISEL-positive cells, but both label-positive localizations were closely related to each other. In addition, they were detected more often in epithelial cells cultured with low testosterone concentrations. By the QF-DE method, chromatin fibers were found to be broken in spotty parts of apoptotic nuclei. We could control the concentration of testosterone in culture medium and detect the appearance of Fas antigen in cultured prostatic epithelial cells, followed by apoptotic changes. So, Fas and Fas-ligand system is one candidate for apoptosis in the prostate glands, depending on removal of hormonal testosterone.

Three-dimensional ultrastructure of apoptotic nuclei in rat prostatic epithelial cells revealed by a quick-freezing and deep-etching method.

To clarify intranuclear apoptotic changes, we have investigated chromatin organization in apoptotic nuclei of castrated rat prostatic cells by a quick-freezing and deep-etching (QF-DE) method. The ventral prostates taken from intact and castrated adult male rats were investigated by light microscopy, in situ end-labeling (ISEL) technique, conventional electron microscopy, and the QF-DE method. In control nuclei, the chromatin fibers were uniformly distributed and formed a network structure. In apoptotic nuclei, destruction of such chromatin networks was detected, which was clearly seen by the QF-DE method. Although it first appeared spotty in the apoptotic nucleus, definite destruction of the intranuclear network occurred in the nuclear center at later stages, and broken fibrous structures were condensed along the nuclear margin. The ISEL technique was applied to the QF-DE method. Localization of damaged DNA fragments could three-dimensionally be detected on replica membranes. Intranuclear chromatin organization in apoptotic cell death of rat prostates was observed by the QF-DE method. We could examine early-stage apoptotic nuclei at an electron microscopic level, which would not be clarified by other conventional methods.

Morphological studies of glomeruli in obstructive kidneys by confocal laser scanning microscopy and quick-freezing replica method.

Morphological changes of glomeruli in obstructive kidneys were studied by using confocal laser scanning microscopy (CLSM), and quick-freezing and deep-etching (QF-DE) method. Twenty-one rabbits were divided into three groups, consisting of control, 6-hr bilateral ureteral obstruction (BUO) and 24-hr BUO. In the experimental groups, the attenuation of cell bodies, the lengthening and stretching of major processes, the cystic formation in the cytoplasm and the fusion of foot processes were observed on conventional ultrathin sections. These changes in the 24-hr BUO group were more clearly observed than those in the 6-hr BUO group. By the CLSM, cell bodies and foot processes of podocytes in the experimental groups were more intensely immunostained with anti-alpha-tubulin antibody and phalloidin-FITC. By the QF-DE method, cytoskeletons in the podocyte cell bodies and major processes were composed of numerous intermediate filaments, but distinct changes of actin filaments and microtubules were not observed in the control and experimental groups. Considering the physiological changes in BUO, the mechanical stress appeared to be brought about by hemodynamic factors rather than the change of intratubular pressure, resembling the morphological changes in experimental animals with hyperfiltration and the homeostatic adaptation of podocytes under the BUO condition.

Ultrastructural Study of Anionic Sites in Glomerular Basement Membranes at Different Perfusion Pressures by Quick-Freezing and Deep-Etching Method

The ultrastructures of anionic sites in rat glomerular basement membranes (GBM) were studied at different perfusion pressures of 100, 150, 200 or 250 cm H₂O by a quick-freezing and deep-etching (QF-DE) method, in addition to conventional fixation methods, using polyethyleneimine (PEI) as a cationic tracer. By the QF-DE method, three-dimensional ultrastructures at each pressure were observed more clearly than those seen on conventional ultrathin sections. When the perfusion pressures were changed from low levels to higher ones, the total thickness of GBM with anionic sites became gradually thinner. Many PEI particles were observed around filaments, not only in the laminae lucidae, but also in the lamina densa. These findings indicated the existence of anionic sites in both laminae lucidae and lamina densa of GBM. The numbers of PEI particles in the lamina rara externa were counted on conventional ultrathin sections for morphometric analyses. The numbers per unit length of GBM were significantly decreased at higher perfusion pressures (200 and 250 cm H₂O) than those seen at both normal (150 cm H₂O) and lower (100 cm H₂O) pressures. It is concluded that the ultrastructures of anionic sites in the GBM may be changed in vivo, depending on the hemodynamics in the glomerular capillary.

Proteoglycans in articular cartilage revealed with a quick freezing and deep etching method.

To clarify the three dimensional ultrastructure of proteoglycans, and their relationship with other matrix components in articular cartilage. Specimens from rat femoral heads were examined using three techniques: (1) Histochemical staining with cationic polyethyleneimine (PEI), using a pre-embedding or a postembedding method. Some tissues were pretreated with chondroitinase ABC or hyaluronidase. (2) Quick freezing and deep etching (QF-DE). Some specimens were fixed with paraformaldehyde and washed in buffer solution before quick freezing; others were frozen directly. (3) Ultrathin sections were studied after conventional preparation. Proteoglycans were observed as aggregated clumps with PEI staining by the pre-embedding method, but as fine filaments by the postembedding method. They were lost with enzyme digestion; this was also demonstrated by the QF-DE method. The ultrastructure was well preserved by the QF-DE method when fixation and washing procedures were included, but not without these procedures. A fine mesh-like structure was connected to the cell membrane in the pericellular matrix. Filamentous structures suggestive of aggrecans were observed among collagen fibrils. They had side chains, approximately 50 nm in length, which branched from the central filaments at intervals of 10-20 nm, and were occasionally linked to other structures. Many thin filaments were also attached to the collagen fibrils. The QF-DE method incorporating paraformaldehyde fixation and buffer washing procedures revealed three dimensional, extended structures suggestive of proteoglycans.

Developmental studies of dystrophin and other cytoskeletal proteins in cultured muscle cells

We studied the developmental changes of localization of dystrophin and other cytoskeletal proteins, especially actin, spectrin and dystrophin related protein (DRP) using immunocytochemistry and quick-freezing and deep-etching (QF-DE) method. In developmental studies of mouse and human muscle cultures, some myoblasts had positive-reactions to spectrin, DRP, and F-actin, but not dystrophin. In aneurally cultured myotubes, dystrophin, DRP, and spectrin were localized diffusely in the cytoplasm and later in discontinuous patterns on the plasma membrane, when myotubes became mature. Spectrin and DRP had more positive reactions in immature myotubes, compared with those of dystrophin. In some areas of myotubes, dystrophin/spectrin and spectrin/actin were localized reciprocally. In innervated cultured human muscle cells, dystrophin and DRP were localized in neuro-muscular junctions, which were co-localized with clusters of acetylcholine receptors. By using the QF-DE method, dystrophin was localized just underneath the plasma membrane, and closely linked to actin-like filaments (8-10 nm in diameter), most of which were decorated with myosin subfragment 1. In actin-poor regions, spectrin was detected as well-organized filamentous structures in highly interconnected networks with various diameters. DRP was distributed irregularly with granular appearance inside the cytoplasm and also under the plasma membrane in immature mouse myotubes. Our present studies show that dystrophin, spectrin, and DRP are localized differently at the developmental stages of myotubes. These results suggest that dystrophin, spectrin, and DRP are organized independently in developing myotubes and these cytoskeletal proteins might play different functions in the preservation of plasma membrane stability in developing myotubes.

Three-dimensional study of epithelioid cells by a quick-freezing and deep-etching method in muramyl dipeptide-induced granulomas.

The three-dimensional ultrastructure of epithelioid cells was studied by the quick-freezing and deep-etching (QF-DE), as well as the freeze-substitution (QF-FS) methods. The granulomas were induced in rats by injecting muramyl dipeptide (MDP) into the hind footpads. At 3 weeks after the injection, the footpads were perfused with a fixative, excised, and quickly frozen to prepare the replica membranes. Some unfixed footpads were also quickly frozen and freeze-substituted. Dense networks of intermediate filaments, connected with the nuclei, mitochondria and other vesicular cell organelles, were observed throughout the cytoplasm of epithelioid cells by the QF-DE method. A few actin filaments were located in filopodia and just beneath the cell membranes. Interdigitation of the cell membranes between adjacent cells was clearly demonstrated by the QF-FS method and clathrin-coated pits were identified at the base of interdigitating filopodia. In addition, the exact moment of fusion between endosomes and lysosomes was ascertained by the same method. These results suggest that the cytoskeletal organization of epithelioid cells resembles that of epithelial cells rather than actively motile macrophages.

Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of Cryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method. C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10(7) cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells of C. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure of C. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10-13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions by C. neoformans.

Ultrastructural study of normal rat glomeruli by the quick-freezing and deep-etching method.

The ultrastructural features of isolated normal rat glomeruli were investigated using a new splitting technique and the quick-freezing and deep-etching (QF-DE) method. Examinations were also made of in vivo normal rat glomeruli by the QF-DE method to visualize the glomeruli under near natural conditions. Freeze-fractured capillary loops were observed from the capillary lumen or the urinary space. Foot processes were found to be freeze-fractured horizontally, obliquely or vertically to the glomerular basement membrane (GBM), and a glomerular slit diaphragm, which consisted of sheet-like, ladder-like and zipper-like substructures, was clearly identified. The GBM was classified into three zones, which might correspond to those seen in ultrathin sections, including the lamina densa and laminae rarae externa and interna. The lamina densa was composed of a meshwork filled with fine particles, which might represent products of insoluble proteins. In the laminae rarae, filamentous structures consisting of irregular networks could be observed connecting glomerular endothelial or epithelial cells with the lamina densa. Furthermore, the above findings were obtained from investigations at various consecutive levels of freeze-fractured capillary loops from various angles. The three-dimensional ultrastructure of the glomeruli could be demonstrated at high resolution by the QF-DE method.