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Vital fluorescent staining technique for microspores of Brassica napus. 甘蓝型油菜小孢子生命荧光染色技术。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009105615
E B Swanson, S A Yarrow, M P Coumans, L R Erickson

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.

用三种染色法观察了早期小孢子胚发育的个体发生过程。3,3'-二乙基氧基二碳氰碘化染色剂,先前被报道为线粒体特异性染色剂,也被观察到显示了发育中的甘蓝型油菜小孢子的外壁。当与Tinapol 5 BM(一种纤维素细胞壁染色剂)联合使用时,它提供了高对比度,并有助于鉴定具有胚胎发生潜力的小孢子。Hoechst 33342是一种核染色剂,单独使用或与其他染色剂中的一种或两种结合使用,可用于突出小孢子的核发育阶段。本文描述了使用这些材料对外壁、细胞壁/内壁和细胞核进行特异性染色的程序,从而允许在小孢子衍生胚胎发育的早期阶段跟踪它们的命运。
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引用次数: 3
Quantitative ultrastructural immunocytochemistry using a computerized image analysis system. 定量超微结构免疫细胞化学使用计算机图像分析系统。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009105618
S Eneström, B Kniola

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultrathin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au7, Au11, Au17). The time of pretreatment of sections with H2O2 or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.

用免疫金标记法间接检测了人垂体腺瘤细胞分泌颗粒的激素表位。利用计算机图像分析系统测量了分泌颗粒上不同免疫球蛋白-金复合物与抗生长激素抗体的特异性结合。这有助于评估三种不同平均粒径(Au7, Au11, Au17)的金颗粒与靶颗粒的优先结合。发现用H2O2或缓冲液预处理切片的时间对染色有很大影响。腺垂体分泌颗粒表面呈山状突起,扫描电镜结果可能与腺垂体分泌颗粒表面免疫标记分布不均匀有关。
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引用次数: 14
A new technique for C-banding. c带的新技术。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009105609
J D Bugallo, R Teresa, M Vallejo
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引用次数: 1
Visual enhancement of myelinated tissues in the central nervous system of the rat using Sudan black B. 苏丹黑B对大鼠中枢神经系统髓鞘组织的视觉增强作用。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009139928
K R Olson, R K Traub
Abstract : This report investigated the quantitative distribution of radio labelled compounds in the central nervous system of the rate using autoradiographic techniques. To complement our autoradiographic images, we searched for a suitable fiber stain delineating myelinated structures using fresh frozen tissue. Surprisingly, the majority of staining procedures were exclusive to fixed tissues. These techniques applied to fresh frozen brain tissue produced poor differentiation of myelinated and nonmyelinated areas and were generally unsatisfactory. Therefore, we preformed a series of studies using lipid soluble dyes such as oil red O, Luxol fast blue, hematoxylin and Nile blue in an effort to enhance differentiation of myelin. We found Sudan black B to be superior to enhance differentiation of myelin. We found Sudan black B to be superior for our purpose.
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引用次数: 4
Modified Golgi method for whole rat brain. 改进的大鼠全脑高尔基法。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009139929
E R Greer, C K Jen
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引用次数: 0
Cement line staining in undecalcified thin sections of cortical bone. 骨皮质未钙化薄切片的水泥线染色。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009108065
S D Bain, T M Impeduglia, C T Rubin

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

一种在薄的、未钙化的皮质骨横切面上显示水泥线的技术已经发展起来。皮质骨样品经过处理,并在甲基丙烯酸甲酯塑料中嵌入未钙化。在3-5微米处切片后,将横截面转移到玻璃载玻片上并压平10分钟。皮质骨切片在1%甲苯胺蓝溶解于0.1%甲酸的新鲜溶液中自由漂浮染色20秒。切片在丁醇中脱水,在二甲苯中清除,并用Eukitt培养基安装。逆转线在浅蓝色基质上呈现出细长的扇形深蓝色线条,而骨形成阻止线则较粗,轮廓光滑。这种技术保留了细胞细节、类骨分化和荧光标记。结果证明了水泥线一步染色方法的适用性,这将有助于评估骨重塑活动的薄切片未钙化皮质骨。
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引用次数: 31
A simple method for improved visualization of the lamellated structure of cutinized and suberized plant cell walls by electron microscopy. 一个简单的方法,提高可视化的层状结构的角质化和隐性植物细胞壁的电子显微镜。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009108068
H G Heumann

By treating ultrathin sections with H2O2 prior to normal uranyl acetate and lead citrate staining, a strong increase in contrast of cutinized and suberized plant cell walls can be achieved.

在正常的醋酸铀酰和柠檬酸铅染色之前,用H2O2处理超薄切片,可以实现角质化和钝化植物细胞壁的对比度的强烈增加。
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引用次数: 6
A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC. 一种同时鉴定骨骼肌纤维类型I、IIA、IIB和IIC的渐变色染料- atp酶法。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009105613
R W Ogilvie, D L Feeback

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C, followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, IIA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.

在不同类型的人类骨骼肌纤维的选择性pH值下,肌纤维atp酶活性的定量差异的组织化学证明是其分化最广泛使用的技术。该反应的基础是由肌纤维ATP酶从ATP中分离出的不溶性无机磷酸盐盐沉积,然后用可溶性较低的显色盐取代磷酸盐。Doriguzzi和他的同事用异色染料证明了不同纤维类型中磷酸盐沉积的数量差异。在常规atp酶组织化学和蓝色A或甲苯胺蓝染色后,对低atp酶活性(低磷酸盐含量)的纤维进行偏色染色,而高atp酶活性(高磷酸盐含量)的纤维进行正色染色,颜色强度与不溶性磷酸盐的含量成正比。在水溶液中过度洗涤或在乙醇中常规脱水后,异色很容易丢失,因此纤维类型区分减弱。据报道,该技术的一项关键改进是在室温(22-24℃)下(而不是通常的37℃)将人体骨骼肌冷冻切片在含atp的培养基中孵育,然后修改洗涤和脱水方案。通过这些改进,可以在单个切片中快速可靠地识别四种人类骨骼肌纤维类型(I、IIA、IIB和IIC),从而避免了对连续切片进行检查的需要。即使在黑白照片中,颜色的区分也允许纤维类型的识别。
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引用次数: 122
A simple method for the cultivation of cell monolayers for electron microscopy studies. 一种用于电镜研究的细胞单层培养的简单方法。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009105610
R M Pintó, J Jofre, A Bosch
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引用次数: 2
Electron microscopic identification and morphologic preservation of enriched populations of lung cells isolated by laser flow cytometry and cell sorting: a new technique. 激光流式细胞术和细胞分选分离的肺细胞富集群体的电镜鉴定和形态学保存:一项新技术。
Pub Date : 1990-01-01 DOI: 10.3109/10520299009108066
D P Penney, J F Leary, R A Cooper, A Paxhia

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).

越来越需要验证通过激光流式细胞术和荧光激活细胞分选(FACS)富集的细胞亚群的身份。当从整个器官或组织中分离的细胞亚群具有相似的特征(例如,大小,粒度,染色)时,光,相对比或荧光显微镜可能无法提供足够的分辨率来准确识别分离的细胞,并且许多流式细胞术参数(例如,活力,荧光)要求细胞在细胞横切激光束的分析点上是活的。在一些研究中,通过荧光显微镜鉴定为高度富集亚群的细胞通过电子显微镜发现含有其他细胞类型的显著群体。一种技术,固定在流动(FIF),已经发展起来,以增加能力相关联的形态学和激光分析细胞亚群。用固定液代替鞘液,允许在激光束分析活细胞后立即开始固定。这种新方法提高了恢复细胞亚群的细胞结构保存,以便通过透射或扫描电子显微镜进行评估。本报告介绍了应用该方法更准确地识别远端肺细胞亚群的结果,特别是II型肺细胞、Clara细胞和肺巨噬细胞。该方法的修改被用于从体外培养的小鼠肺成纤维细胞中分离成纤维细胞亚群,并且该方法被用于确定培养成纤维细胞对其他排列(例如x射线照射,细胞因子)的反应。
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引用次数: 3
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Stain technology
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