介绍一种半薄切片定位样品的方法

介绍了一种半薄切片定位样品的方法。此法与前人的方法不同之处在于位于载玻片之上被重新包埋于塑料环内的切片与载玻片的分离真人处步骤是“切片被包埋聚合好后,立即从６０￣６５℃温箱内被转入冰箱冷冻室中（－１８℃）放置５￣１０分钟。然后,将载玻征从冷冻室中取出,轻推塑料环,即可使包埋在塑料环内的切片与载玻片分离。这一方法成功地解决了样品中靶细胞的发育时期确定和样品丢失问题。而且还有简单、易操作和成功率高等优     

一种冰冻切片漂片法免疫反应后再用石蜡切片的方法

本文采用组织冰冻切片５０μｍ进行双重标记的免疫组织化学漂片染色后,将其贴在塑料包埋盒内,脱水,经石蜡包埋切为１－３μｍ的薄片贴于载玻片上,干燥后脱水透明封片,最后进行切片观察,免疫阳性产物被准确定位于所观察的细胞或纤维上,得到满意的结果。为免疫组织化学形态学研究工作提供了新的手段。     

少量贴壁培养细胞电镜原位包埋方法的探讨

该文探讨了对少量贴壁培养细胞较易操作且能保存较好超微结构的透射电镜样品包埋的方法。将Hela细胞分为三组:(1)不使用环氧丙烷,将树脂胶囊直接倒扣包埋于塑料培养皿;(2)不使用环氧丙烷,将细胞爬片倒扣包埋于胶囊;(3)使用环氧丙烷并将细胞爬片倒扣包埋于胶囊。将三组带有细胞的树脂胶囊进行超薄切片,电镜观察后发现,第一种方法包埋简便,超薄切片上无细胞缺失孔洞,且超微结构保存较好。     

火棉胶包埋组织切片及冰冻切片的连续切片与染色

近年来为了教学和研究所用之切片标本,曾广泛使用火棉胶包埋组织切片和冰冻切片法,这两种方法虽然它是有比石腊包埋组织切片稍厚一点,但它的优点是远超过了缺点。组织用石腊包埋,在组织经过不同浓度酒精脱水后,须用氯仿或二甲苯透明,再置于58℃温箱石腊中几个小时。以上透明剂具有使组织收缩变小,在经过这样高温之下,组织增加它由收缩而扭转和变形,纤维曲折,细胞及所含物质缩小,折光率增强,组织内所含之尖脂脂亦易于被以上透明剂所溶解,致染色不佳。作为观察和研究组织形态是不够美满的。因为石腊包埋组织制作切片有以上之缺点,     

用于光学显微镜研究的塑料半薄切片

本文介绍了以环氧树脂为包埋介质的用于光学显微镜的塑料半薄切片的制备技术和部分实验结果。叙述了固定、脱水、渗透、包埋、聚合、切片、染色及封片各程序。作为对石蜡切片技术的补充和发展。塑料半薄切片能充分发挥光学显微镜的分辨能力,能观察到许多在石蜡切片上看不清或看不到的细胞内部结构,如:花粉的外粉壁,萌发孔;细胞的微核,液泡和液泡问的原生质丝等。可用同一包埋材料在半薄切片基础上进行超薄切片,所以半薄切片技术是一种把光学显微镜水平的研究和电子显微镜水平的研究联系在一起的一种过渡性技术。因此,它无论对植物学工作者或其它生物学工作者都是很有用的一项技术。     

塑料薄切片技术

光学显微镜术观察用的薄切片,可用各种塑料包埋剂,如乙二醇甲基丙烯酸酯(glycolmethacrylate)和环氧树脂类(epoxy resins)包埋。这些塑料包埋剂经凝固后,很容易被切成1—2微米的薄切片,比传统的石蜡切片要薄得多;而且染色时无需经过脱蜡手续,便可     

一种重包埋方法的应用

目前普遍使用的简单定向方法,是先在包埋对使样品定向,以后再将聚合好的包埋块切一张大面积的1—2微米的厚切片,经光学显微镜检查找出所需要的部位,做好标记,然后对照厚切片修整样品再作超薄切片。Grimley 曾报道一种简单的重包埋方法,将包埋的组织块简单地切成一张1—8微米的厚片,贴在盖玻片上染色后,在光学显微镜下找出需要进行电镜观察的部位,标上记     

冰冻切片技术在植物显微结构和组织化学中的应用

介绍了冰冻切片法研究植物显微结构和组织化学的一般程序。结果表明,冰冻切片过程简单有效且图版清晰,在1d内可获得高质量图片,解决了利用普通石蜡切片观察植物样品需要进行脱水、浸透、包埋操作且耗时长的问题,在植物显微结构和组织化学研究中具有广阔的应用前景。     

一种简单快速植物组织冰冻切片方法

比较不同冷冻方法对植物细胞超微结构的影响,结果表明:直接包埋法处理的植物细胞超微结构保存较好,而液氮冷冻处理的植物细胞内膜系统损伤严重.建立了一种直接包埋冷冻和适当回温相结合的方法,不仅可以制作出植物细胞基本结构保存完整的组织切片,而且避免了使用冰冻保护剂的弊端.其操作程序是:样品固定→冰冻与包埋→适当回温→快速切片→展片→染色.此法制作的切片可进行不同的染色和组织细胞化学测定,具有操作简便,易于推广的特点.     

介绍一种超薄切片中细胞定位的薄层包埋方法

电子显微技术中,对研究材料中的特定组织或细胞的定位在超薄切片时是最困难的一步。曾提出的用还氧树脂厚切片重新包埋的方法以及类似的厚切片重新粘贴法在克服这一困难上有很好的效果。对于单细胞或分离的原生质体,以及花粉和萌发的花粉管等材料,电镜样品的制作常用离心进行材料的收集、固定、脱水和渗透等步骤,并按寻常的方法     

Embedding Stained Mammary Glands in Plastic

A simplified method of embedding stained mammary spreads of small mammals in plastic (Selection) is presented. Two standard 50 × 75 × 1 mm. glass slides, separated by 2 narrow glass strips of similar thickness, are used to form the embedding chamber. The glands are stained in toto in alum-carmine, dehydrated, defatted, infiltrated with uncatalyzed plastic and embedded in catalyzed plastic. After baking and cooling, the glass chamber separates readily and provides a thin square slide of plastic suitable for low-power microscopic examination, projection, and filing.     

The Rapid Preparation of Frozen Tissue Sections

The essential feature of this procedure involves the rapid freezing of the tissue following excision and keeping it frozen until the desired chemical or fixative has been applied. For freezing, either carbon dioxide or liquid air is used, as desired. The microtome knife is thoroughly cooled by taping blocks of dry ice to its surface. The cut sections, still frozen, are manipulated by a camel's hair brush so that they lie flat upon the knife. They are then transferred to a slide by a special section lifter. This has the form of a double-bottomed scoop packed with dry ice. Thus the section remains frozen while it is transferred to a clean microscope slide held at an angle above a Coplin jar of the desired reagent. The sections must be immersed just prior to melting. They curl and do not adhere to the slide if still rigidly frozen, and are distorted if immersed after melting.

With this technic sections showing a minimum of cellular distortion may be obtained. Consequently, it facilitates the use of many cytological technics, chemical tests, and enzymatic studies, such as the Gomori technics, on a variety of tissues.     

Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections

We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR.     

Freezing by Liquid Carbon Dioxide in Making Slides Permanent

The expansion of liquid CO₂ may be employed in a quick-freeze method for making aqueous slide preparations permanent. An apparatus is described for this purpose which could be duplicated satisfactorily by cutting a 22mm square hole in the top of a standard freezing microtome specimen holder. The edges should be filed smooth to provide a flat surface for the slide to rest on, and clamps added to keep the slide in place while freezing. Once the slide is frozen, the cover slip may be readily removed, leaving practically all of the tissue on the slide. Following simultaneous thawing and dehydration of the slide in 95% alcohol, covering is done with Diaphane or Euparal and a clean, dry cover slip.     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.     

Controlling the freezing process: a robotic device for rapidly freezing biological tissues with millisecond time resolution

A robotic cryogenic device was developed which allows freezing of thick biological tissues with millisecond time resolution. The device consists of two horizontally oriented hammers (pre-cooled with liquid N(2)) driven by two linear servo-motors. The tissue sample is bathed in Ringers contained in a chamber which drops rapidly out of the way just as the hammers approach. A third linear motor is vertically oriented, and permits the rapidly dropping chamber to smoothly decelerate. All movements were performed by the three motors and four solenoids controlled by a PC. Mechanical adjustments, that change the size of the gap between the hammers at the end position, permit the final thickness of the frozen tissue to be varied. Here we show that the freezing time increased with the square of the final thickness of the frozen bundle. However, when bundles of different original thicknesses (up to at least 1mm) were compressed to the same final thickness (e.g., 0.2mm), they exhibited nearly equal freezing times. Hence, by being able to adjust the final thickness of the frozen bundles, the device not only speeds the rate of freezing, but standardizes the freezing time for different diameter samples. This permits the use of freezing for accurate determination of the kinetics of cellular processes in biological tissue.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

A simplified technique for the short-term tissue culture of rabbit corneal cells

Summary A technique for the short-term culture of pure populations of rabbit corneal endothelial and epithelial cells has been developed. Rabbit corneas were placed on concave agarose surfaces, treated briefly with a solution of trypsin and ethylenediamine tetracetic acid, and transferred, either epithelial cell surface or endothelial cell surface down, to microscope slide culture chambers. Within 6 to 12 h the epithelial cells or endothelial cells attached to the slide chamber surface and the cornea was removed, leaving behind a pure population of cells which spread out and grew to fill the surface of the slide chamber. This technique provides a simple and economic means for the reproducible initiation of primary cultures of rabbit corneal epithelial and endothelial cells for us in a variety of experiments. This study was supported in part by Public Health Service grants EY03150, EY02580, and EY02377 from the National Eye Institute, National Institutes of Health, Bethesda, MD, and a Foreign Fellowship (Dr. Xie) from Research to Prevent Blindness, Inc., New York, NY.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.