On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.     

"Contractile interstitial cells" in pulmonary alveolar septa: a possible regulator of ventilation-perfusion ratio? Ultrastructural, immunofluorescence, and in vitro studies

In the lungs of healthy rats, humans, lambs, and monkeys, about 50% of the alveolar interstitial cells—resembling fibroblasts—contain bundles of fibrils measuring 30–80 Å in diameter. Immunofluorescence studies on frozen sections of rat lung demonstrate that many interstitial cells bind sera containing antiactin antibodies. On account of these two sets of findings and our additional in vitro studies suggesting alveolar tissue contraction due to hypoxia or epinephrine, we postulate that the alveolar septa contain contractile cells different from that of smooth muscle. For these cells we propose the name of "contractile interstitial cells." Such cells lie within the thick portion of the air-blood barrier and around the pre- or postcapillary vessels. Hence it is possible that they play a role in the autoregulation of ventilation/perfusion (V/Q) ratio, particularly in hypoxic pulmonary hypertension. These findings, demonstrating a contractile system other than bronchial and arterial smooth muscle, suggest that the alveolus should no more be conceived as a passive "organ."     

Electron Microscopic Study of Membranes and Walls of Bacteria and Changes Occurring During Growth Initiation

Thin sections of stationary-phase Streptococcus lactis cells showed that the wall and membrane are 20 and 7 nm thick, respectively. Whole cells were examined by negative staining with ammonium molybdate and by shadowing. On air-drying of whole cells, the membrane pulled away from the wall revealing adhesions between these organelles. Adhesions could not be seen after subculture of the stationary-phase cells into complex media or into solutions containing glucose, KCl, and CaCl(2) in tris(hydroxymethyl)aminomethane buffer. The adhesions were also observed in stationary-phase cells of other gram-positive bacteria. Fractured freeze-etched cells of S. lactis had a smooth outside surface, but the inside of the wall (or outside of the membrane) had a regular structure, repeating at 10 nm, which could correspond to the adhesions observed in the negatively stained air-dried cells. Freeze-etching also revealed holes in the outside wall which had the shape of inverted truncated cones. The outside diameter of the cone was 60 nm, and the diameter on the inside surface of the wall was 20 nm. The membrane had upstanding plugs, 20 nm in diameter, which could fill the holes in the wall.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

A combined silver and acetylcholinesterase method for staining intramuscular innervation.

A combined acetylcholinesterase and silver stain for demonstrating the intramuscular innervation of fresh frozen tissue is described. Intramuscular nerves, subterminal axons, and motor end plates are simultaneously stained brown or black with minimal staining of connective tissue and muscle fibers in longitudinal sections 30-100 mu thick. The method has been applied to fetal and adult rat, porcine, and bovine skeletal muscle. Antemortem and postmortem tissue samples stained equally well. The method facilitates simultaneous appreciation of morphological alterations in nervous and muscular tissues; in clinical and research laboratories alike it is of value when muscle abnormalities which may be related to disorders of nervous origin are studied. Compared with other published procedures this method has shorter time requirements, uses fresh frozen tissue, and displays superior staining characteristics.     

A Combined Silver and Acetylcholinesterase Method for Staining Intramuscular Innervation

A combined acetylcholinesterase and silver stain for demonstrating the intramuscular innervation of fresh frozen tissue is described. Intramuscular nerves, subterminal axons, and motor end plates are simultaneously stained brown or black with minimal staining of connective tissue and muscle fibers in longitudinal sections 30-100 μ thick. The method has been applied to fetal and adult rat, porcine, and bovine skeletal muscle. Antemortem and postmortem tissue samples stained equally well. The method facilitates simultaneous appreciation of morphological alterations in nervous and muscular tissues; in clinical and research laboratories alike it is of value when muscle abnormalities which may be dated to disorders of nervous origin are studied. Compared with other published procedures this method has shorter time requirements, uses fresh frozen tissue, and displays superior staining characteristics.     

LOCUS AND STATE OF AGGREGATION OF MYOSIN IN TISSUE SECTIONS OF VERTEBRATE SMOOTH MUSCLE

Structures with the characteristics of molecular myosin were identified by electron microscopy in tissue sections of vertebrate smooth muscle. No thick filaments of myosin were found regardless of preparative procedures, which included fixation at rest and in contraction, glycerine extraction, and storage at low pH prior to fixation. Absence of thick myosin filaments and presence of what appear to be myosin molecules is in accord with conclusions based on X-ray diffraction (3, 12) and birefringence data (4) from living smooth muscles at rest and in contraction. Explanations are provided for appearances thought by others (6, 20, 21) to represent thick myosin filaments. Our present observations are in accord with the model for smooth muscle contraction which we have previously proposed (1).     

Electron tomographic analysis of frozen-hydrated tissue sections

Electron tomography of frozen-hydrated tissue sections enables analysis of the 3-D structure of cell organelles in situ and in a near-native state. In this study, 160-200-nm-thick sections were cut from high-pressure frozen rat liver, and improved methods were used for handling and mounting the sections. Automated data collection facilitated tilt-series recording at low electron dose (approximately 4000 e(-)/nm(2) at 400 keV). Higher doses (up to 10,000 e(-)/nm(2)) were found to increase contrast and smooth out surface defects, but caused section distortion and movement, with likely loss of high-resolution information. Tomographic reconstruction showed that knife marks were 10-40 nm deep and located on the "knife face" of the section, while crevices were 20-50 nm deep and found on the "block face." The interior of the section was normally free of defects, except for compression, and contained useful structural information. For example, the topology of mitochondrial membranes in tissue was found to be very similar to that in frozen-hydrated whole mounts of isolated mitochondria. In rare cases, a 15-nm banding pattern perpendicular to the cutting direction was observed in the interior of the section, most evident in the uniformly dense, protein-rich material of the mitochondrial matrix.     

Combination of lamin immunocytochemistry and in situ hybridization for the analysis of chromosome copy numbers in tumor cell areas with high nuclear density

We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

Ultrastructural investigations on the anterior adductor muscle of a brachiopoda, Lingula unguis

The brachiopoda, Lingula unguis, has a pair of anterior adductors located in the center of the shell. Each muscle consists of an opaque and a translucent portion which is constructed of smooth and obliquely-striated muscle respectively. According to our ultrastructural observations, the opaque portion seems to have two types of cells. They differ only in the diameters of their thick myofilaments. The fine structure of their cell organelles resembles each other. We measured the diameters of the thick myofilaments in each type of cell to distinguish between the two cell types. About 500 measurements of myofilament diameters were made for each type of cell and statistically analyzed. For one type of cell, the distribution of diameters of the thick myofilaments fit a normal distribution curve with a peak at 37-60 nm. The distribution of diameters of the thick myofilaments for the other type fit a curve in which two normal distribution curves having peaks at 37-60 and 75-97 nm respectively partially overlapped. According to these results, we suggest that the opaque portion contains two types of cells, each having a different distribution of thick myofilament sizes.     

Ultrastructure of muscle cells in the adductor of the boring clam Tridacna crocea

The ultrastructure of the adductor muscle of the boring clam (Tridacna crocea) was investigated. The adductor was composed of opaque and translucent portions. The opaque portion contained smooth muscle cells; the translucent portion contained obliquely striated cells. Smooth muscle cells were classified, according to the statistically analyzed diameters of their thick myofilaments, into two types, S-1 and S-2. S-1 cells had thick myofilaments, 50–60 nm in diameter. S-2 cells had thick myofilaments of two sizes, about 55–65 nm and 85–100 nm in diameter, respectively. Obliquely striated muscle cells in the translucent portion were also classified into two types: O-1 cells, with thick myofilaments 30–35 nm in diameter, and O-2 cells, with myofilaments of 50–60 nm.     

Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography.

Automated electron tomography is shown to be a suitable means to visualize the shape of phospholipid vesicles embedded in vitrified ice. With a slow-scan charge-coupled device camera as a recording device, the cumulative electron dose needed to record a data set of 60 projections at a magnification of 20,000X can be kept as low as 15 e-/A2 (or 1500 electrons/nm2). The membrane of the three-dimensionally reconstructed vesicles is clearly visible in two-dimensional sections through the three-dimensionally reconstructed volume. Some edges indicating a polygonal shape of the vesicles, frozen from the gel phase, are also clearly recognized. Because of the presently limited tilt angle range (+/- 60 degrees), the upper and lower "caps" of the vesicles (representing about 35% of the surface of the ellipsoidal particles) remain invisible in the three-dimensional reconstruction.     

Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin

Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.     

Contraction-free, fume-fixed longitudinal sections of fresh frozen muscle

Contraction damage occurring when longitudinal frozen sections of fresh unfixed muscles are thawed on microscope slides has limited histological examination of this tissue mainly to cross sections. Longitudinally oriented sections are advantageous for investigating properties that vary along the length of the muscle fibers. A fume fixation technique has been developed for preventing contraction of thick longitudinal frozen sections. The technique is compatible with histochemical staining of enzymes.     

Ultrastructural differences between the anterior and posterior adductors of the strawberry cockle, Fragum unedo

Adductors of Fragum unedo were observed ultrastructurally and their muscle cells were classified according to the statistically analyzed diameter of their thick myofilaments. Two types of smooth muscle cells were observed in the opaque portion of the anterior adductor: A-type cells containing thick myofilaments of about 46 nm in diameter and B-type cells having 62 nm thick myofilaments. The posterior adductor was also composed of two kinds of cells: the B-type cell, which had thick myofilaments of about 67 nm in diameter, and the C-type, containing thick myofilaments of 90 nm. Two types of oblique-striated cells were commonly recognized in the translucent portions of anterior and posterior adductors. Our observations thus indicate that the posterior adductor generally consists of cells which have thicker myofilaments than the ones of the anterior adductor.     

Ultrastructurally different muscle cell types in Eisenia foetida (Annelida,Oligochaeta)

Muscles in the body wall, intestinal wall, and contractile hemolymphatic vessels (pseudohearts) of an oligochaete anelid (Eisenia foetida) were studied by electron microscopy. The muscle cells in all locations, except for the outer layer of the pseudohearts, are variants of obliquely striated muscle cells. Cells comprising the circular layer of the body wall possess single, peripherally located myofibrils that occupy most of the cytoplasm and surround other cytoplasmic organelles. The nuclei of the cells lie peripherally to the myofibrils. The sarcomeres consist of thin and thick myofilaments that are arranged in parallel arrays. In one plane of view, the filaments appear to be oriented obliquely to Z bands. Thin myofilaments measure 5–6 nm in diameter. Thick myofilaments are fusiform in shape and their width decreases from their centers (40–45 nm) to their tips (23–25 nm). The thin/thick filament ratio in the A bands is 10. The Z bands consist of Z bars alternating with tubules of the sarcoplasmic reticulum. Subsarcolemmal electron-dense plaques are found frequently. The cells forming the longitudinal layer of the body wall musculature are smaller than the cells in the circular layer and their thick filaments are smaller (31–33 nm centrally and 21–23 nm at the tips). Subsarcolemmal plaques are less numerous. The cells forming the heart wall inner layer, the large hemolymphatic vessels, and the intestinal wall are characterized by their large thick myofilaments (50–52 nm centrally and 27–28 nm at the tips) and abundance of mitochondria. The cells forming the outer muscular layer of the pseudohearts are smooth muscle cells. These cells are richer in thick filaments than vertebrate smooth muscle cells. They differ from obliquely striated muscle cells by possessing irregularly distributed electron-dense bodies for filament anchorage rather than sarcomeres and Z bands and by displaying tubules of smooth endoplasmic reticulum among the bundles of myofilaments. © 1995 Wiley-Liss, Inc.     

Thick filaments in vascular smooth muscle

Two sets of myofilaments were demonstrated after incubation of strips of rabbit portal-anterior mesenteric vein under moderate stretch in a physiological salt solution. Thick filaments had a mean diameter of 18 nm and reached a maximum length of 1.4 µm with a mean length of 0.61 µm. In transverse sections, 2.5–5 nm particles were resolved as subunits of the thick filaments. Thin filaments had an average diameter of 8.4 nm and generally conformed to the structure believed to represent actin filaments in smooth and striated muscles. In the areas of maximum concentration there were 160–328 thick filaments/µm² and the lowest ratio of thin to thick filaments was 12:1. Thick filaments were present in approximately equal numbers in vascular smooth muscle relaxed by theophylline, in Ca⁺⁺-free solution, or contracted by norepinephrine. The same preparatory procedures used with vascular smooth muscle also enabled us to visualize thick filaments in guinea pig and rabbit taenia coli and vas deferens.     

Cryo electron tomography of vitrified fibroblasts: microtubule plus ends in situ

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 nm over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5–3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759–765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex.     

Contraction-Free, Fume-Fixed Longitudinal Sections of Fresh Frozen Muscle

Contraction damage occurring when longitudinal frozen sections of fresh unfixed muscles are thawed on microscope slides has limited histological examination of this tissue mainly to cross sections. Longitudinally oriented sections are advantageous for investigating properties that vary along the length of the muscle fibers. A fume fixation technique has been developed for preventing contraction of thick longitudinal frozen aections. The technique is compatible with histochemical staining of enzymes.