Embedding Plant Tissue with Plastic Using High Pressure: A New Method for Light and Electron Microscopy

An embedding technique has been developed to overcome difficulties that confront light and electron microscopists working with so-called “hard-to-embed” plant tissue. The method was originally described for freeze-dried material. It uses a modified Quickfit Rotaflo Valve and low heat to generate high pressure to aid in the infiltration and embedding of tissue with propylene oxide and plastic. The technique is not too cumbersome and requires 6 days from the dehydration step to the end of the polymerization process. Thick sections (1-2 μm) obtained from material prepared by this method stain readily with toluidine blue, and thin sections for the electron microscope stain satisfactorily following standard treatment with uranyl acetate and lead citrate. The thin sections are stable under the beam of the electron microscope. Results indicate that the quality of tissue preservation with this high pressure embedding technique is as good as that observed using standard embedding methods for electron microscopy.     

一种酚氧化酶原组织定位的胶体金标记免疫电镜方法

以亚洲玉米螟Ostrinia furnacalis Guenée5龄幼虫的中肠和体壁组织为材料,采用Epon812常规包埋方法,以作者实验室制备的酚氧化酶原多克隆抗体为一抗、胶体金标记的羊抗鼠IgG为二抗,采用醋酸双氧铀-柠檬酸铅双染色体系,建立一种胶体金标记的特异性强且超微结构保存较好的亚洲玉米螟幼虫体内中酚氧化酶原免疫电镜定位方法。     

In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing

Localization and distribution of proteoglycans within rat growth plate cartilage were investigated by immunoelectron microscopy. By use of a mixture of three monoclonal antibodies directed against chondroitin sulfate chains and of post-embedding staining by protein A-gold, the immunosensitivity and resolution achieved by electron microscopy within tissue processed by high-pressure freezing, freeze-substitution, and low-temperature embedding were compared with those in tissue preserved by three alternative procedures (i.e., mild chemical fixation in combination with either low-temperature embedding or conventional embedding, and high-pressure freezing and freeze-substitution followed by conventional embedding). The loss of matrix components incurred during each stage of high-pressure freezing, freeze-substitution, and low temperature embedding was also determined by measuring the loss of [35S]-proteoglycans from tissue labeled in vivo, and the results compared with previously determined estimates for tissue processed using conventional techniques. Immunosensitivity, determined as the number of gold particles per unit area, was highest in tissue processed by high-pressure freezing, freeze substitution, and low-temperature embedding. Comparable results (with a reduction of only 3-7%) were achieved within tissue preserved by mild chemical fixation followed by low-temperature embedding. In both procedures where conventional embedding was adopted, sensitivity was considerably reduced (by 51% for high-pressure freezing and freeze substitution and by 74% for mild chemical fixation). Loss of matrix components was negligible during all stages of high-pressure freezing, freeze-substitution, and low-temperature embedding. Such information, and that derived from morphological inspection of the various matrix compartments in cartilage processed by high-pressure freezing, freeze-substitution, and low-temperature embedding (J Cell Biol 98:277, 1984), together demonstrate that application of this technique results in successful immobilization of proteoglycans in situ within cartilage matrix. Although loss of proteoglycans from mildly fixed cartilage embedded under low-temperature conditions is minor, morphological examination of this tissue reveals considerable shifting of proteoglycans within matrix compartments. Hence, even though immunosensitivity may be high, resolution is poor. The beauty of the high-pressure freezing, freeze-substitution, and low-temperature embedding technique is that it combines high immunosensitivity with precise localization of matrix components at the molecular level.     

DNA提取的应用与相关技术分析

为了分析影响提取DNA的有关因素,采用标准酚—氯仿抽提法和蛋白酶消化法提取DNA。结果表明,平均每mL全血可提取200 ~ 300μgDNA;新鲜组织及OCT包埋冰冻组织提取DNA,平均每0.2g组织可获得200~300μgDNA;直接将石蜡标本提取物制作模版,PCR扩增良好。从4种组织中均获得较为纯净的DNA;OCT对组织DNA无不良影响。 Abstract:To explore influence factors of DNA extraction,the protease and phenol-chloroform method was used to extract DNA in whole blood,fresh tissues,frozen tissues embedding with OCT and tissues embedding in paraffin.It results that 200~300μg DNA was extracted from 1ml whole blood or 0.2g fresh tissues or frozen tissue embedding with OCT.DNA extracted from paraffin specimen can be directly used in PCR amplification.Purity DNA can be extracted from four kinds of different tissues.OCT hasn't harmful effect on tissue DNA extraction.     

Orienting and Embedding Small Objects in Paraffin

A beaker filled with ice cubes and water is covered with a plate of sheet metal. Over this is placed a paper embedding boat full of molten paraffin, and an inch or two above it is held a 60-watt soldering iron. The height of the soldering iron is then adjusted so that the paraffin at the lower half of the embedding dish becomes semisolid and the upper half stays liquid. By means of warmed forceps, the specimen is transferred to the dish, where it sinks to the top of the semisolid layer. Here, it is oriented by means of a warmed dissecting needle in relation to a pencil mark previously made on one side of the paper boat. One may take as long as he needs to orient the specimen, and several specimens can be oriented one after the other in the same dish.     

Serial Sectioning Techniques for A Modified LKB Historesin

A glycol methacrylate-based plastic that is capable of producing; serial sections has been introduced by LKB. This plastic, provided in the LKB 2218-500 Historesin Embedding Kit, has been tested in our laboratory for its ribbon forming capacity. Various block sizes, concentrations of the softening agent polyethylene glycol 400 (PEG), and tissue types have been examined to determine the optimal conditions for ribbon formation. Although unmodified LKB Historesin is capable of forming ribbons, these ribbons often break. The addition of PEG to the embedding solution enhances ribbon formation. When sectioning with glass knives the best results are achieved with the addition of 0.2 ml of PEG/5.0 ml of embedding medium. A conventional AO rotary microtome can be used to produce ribbons if, in addition to the added PEG (optimal concentration 0.25-0.30 per 5 ml of embedding medium) a thin layer of dental wax is added to the upper and lower surfaces of the block. Ribbons form more easily on microtomes, such as the LKB Historange, that have a retractable specimen arm. If serial sections are to be produced it is very important that the upper and lower faces of blocks be parallel.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Optimization of Whole-Body Zebrafish Sectioning Methods for Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) methods and protocols have become widely adapted to a variety of tissues and species. However, the MSI literature contains minimal information on whole-body cryosection preparation for the zebrafish (ZF; Danio rerio), a model organism routinely used in developmental, toxicity, and carcinogenicity studies. The optimal medium for embedding and cryosectioning a whole organism or soft-tissue specimen for histological examination is a synthetic polymer mixture that is incompatible with MSI as a result of ion suppression. We describe the optimal methods and results for embedding and cryosectioning whole-body ZF for MALDI-MSI. We evaluated 13 distinct embedding media formulations and found a supportive hydrogel with the consistency of cartilage to be the optimal embedding medium. The hydrogel medium does not interfere with MSI data collection, aids in tissue stability, is readily available for purchase, and is easy to prepare and handle during cryosectioning. Additionally, we decreased the matrix cluster interference commonly caused by α-cyano-4-hydroxycinnamic acid by adding ammonium phosphate to the solvent spray solution. The optimized methods developed in our laboratory produced high-quality cryosections, as well as high-quality mass spectral images of sectioned ZF.     

Glycol methacrylate as an embedding medium for bone

A simple and reliable procedure for embedding undecalcified trabecular bone tissue in noncommercial glycol methacrylate (GMA) has been developed. The embedding mixture includes a monomer, methacrylic acid hydroxyethyl ester; a copolymer, methacrylic acid butyl ester; a cross-linker, ethylene glycol dimethacrylate; a catalyst, Luperco; a chemical initiator (N,N-dimethylaniline) and, to avoid excessive elevation of temperature during polymerization, a heat moderator, alpha-terpinene. The appropriate proportions of these components have been selected to give specimens which can be easily sectioned with classical microtomes and which do not swell but spread evenly on a water surface. Since polymerization occurs at -4 C, the method allows demonstration of such enzymatic activities as acid and alkaline phosphatase and carbonic anhydrase. It provides excellent preservation of bone tissue and in studies of bone metabolism allows histomorphometry as well as visualization of fluorescent labeling and radioactive markers. The cost is significantly less than available commercial kits. In our hands glycol methacrylate is at present more useful than methyl methacrylate and is used in our laboratory for routine embedding of bone tissue.     

Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning

Gelatin capsules with rounded ends clipped off and open ends moistened, affixed to a glass slide and sealed with a 15% gelatin solution are used to embed blocks of tissue in plastic. The surface of the slide serves as an orientation plane for structures of the tissue. The plane end of capsules of polymerized plastic containing no tissue is used in embedding frozen tissue sections. The plastic-infiltrated section is flattened against the capsule end under the weight of a 3/4 inch square of plate glass so that larger sections may be cut and surveyed. Embedding cultured cell monolayers grown on coverslips is accomplished in a comparable manner, but the square of plate glass is not needed as a weight. Block-face localization methods depend on the type of material embedded. With blocks of tissue it is achieved by moistening the face with xylene to develop relief. Thin tissue sections are examined by transmitted light, while cell monolayers are stained on the capsule end with methylene blue.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges.

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Oriented Embedding of Single-Cell Organisms

The dexribed technique facilitates oriented embedding of individual cells in various media for both light and electron microscopy. A fixed Specimen is embedded in a small cube of 2% agar at 40 C and subsequently sealed in the desired orientation to a strip of black paper which then serves as a tab for transferring the specimen during dehydrating and embedding procedures. The beveled ends of the strip indicate the exact location of the specimen in the cube. This technique can be employed for the embedding media used in both light and electron microscopy. It ah permits photomicrographs of the whole specimen to be made which can be compared with photomicrographs of individual sections cut from the specimen in a selected plane.     

A Rapid Technique for Preparing Microorganisms for Transmission Electron Microscopy

A rapid and efficient method of preparing microorganisms for transmission electors microscopy is reported. In developing the method Salmonella, streptococcal, ad protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

Stain Historadiography

Fresh bone specimens were dehydrated in acetone and embedded in Ward's Bio-Plastic. Sections 10-15μ thick were cut with a special bone-cutting microtome and subjected first to radiography and then stained with safranin-fast green. The radiograph was made with a Machlett AEG-50-A X-ray tube on a fine-grained, spectroscopic plate; which, after processing, was mounted (dry) on a glass slide adjacent to the stained specimen. By these means, it was possible to make a correlated study of the calcium-bearing parts of the tissue and determine accurately their relationship to the bone itself. The method serves well for investigations of growing bone and for bone that is undergoing pathological involution. It has been named “Stain Historadiography” because it combines a stained specimen with its radiograph.     

云南曲靖张家营一肺鱼齿板

<正> 本文记述的肺鱼齿板是1979年在云南进行野外工作时采获的。标本产自云南曲靖张家营东山中泥盆统曲靖组。登记号V6257 经观察这一标本很可能属于双翼鱼科(Dipteridae),代表一新属、新种。特征一保存不完整的齿板,冠面呈扇形。具9条齿脊,彼此近于平行,脊上具有数目不等的齿突,表面具有琺琅质层。齿谷表面粗糙并缺失琺琅质层。靠近齿板外缘内侧,在齿板冠面上有一浅槽。描述一件保存不完整的左下齿板,仅前侧具脊的部分被保存下来,而后中光滑的台面部分则缺失。齿板中等大小,呈扇形。保存部分的最大长度21毫米,最大宽度16毫     

Extraction of [14C] vitamin A from rat liver and kidney during processing for transmission electron microscopy.

The retention of radioisotope-labeled vitamin A during processing for electron microscopy was investigated using the livers and kidneys of vitamin A deficient rats. [15-14C]Retinol (3muCi/animal) was administered by esophageal intubation to male rats which had been maintained on a vitamin A deficient diet for five or six weeks postweaning. Glutaraldehyde- or osmium-fixed tissue was processed by three methods: a) routine (a graded series of ethanols, propylene oxide and epoxy), b) rapid (75% and 95% ethanol with three changes of epoxy), or c) water-soluble embedding (70% and 80% hydroxypropyl methacrylate). Water-soluble embedding retained the highest percentage of label in the tissue (liver: 96.31%; kidney: 98.68%). Inclusion of osmium tetroxide in the processing sequence and minimal exposure of tissue to lipid solvents were necessary for good retention of labeled vitamin A in tissues.     

A Rigid Illuminator Giving Transmitted Light to Facilitate Microhardness Testing of Calcified Tissues

The structural form of calcified tissues necessitates their examination with transmitted light to identify various morphological features which are not evident with incident illumination alone. To achieve stability for indentation, the specimens must be rigidly mounted. A Leitz Model 514095 base illuminator was fitted with a bottom plate of 3 mm brass for attaching it to the graduated stage and a perforated, 3 mm thick brass top plate, surmounted by a 6 mm thick piece of plate glass. The 25 mm hole in the top plate coincided with a ground glass disc to transmit diffused light through the plate glass to the specimen. Thus, the specimen could be examined on a rigid microscope stage, morphological features identified, and subsequent interpretation of indents made with the usual incident illumination.