New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

New embedding medium for sectioning undecalcified bone.

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

The routine sectioning of undecalcified bone for cytochemical studies

Synopsis A method is described by which serial sections of fresh, undemineralized adult bone can be obtained. Such sections are suitable for cytochemical investigations and can expedite diagnostic histology.     

Evaluation of LR white resin for histology of the undecalcified rat tibia

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.     

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.     

Electron microscopy of dry peanut (Arachis hypogaea L.) seeds crushed for oil removal. Fixation and embedding of anhydrously prepared tissues

Summary Aqueous fixatives caused dry seed tissues to swell; mashed peanuts, crushed to remove oil, swelled even more. Use of anhydrous, organic solvents as vehicles for fixatives enabled maintenance of dimensional stability during fixation of dry seed tissues; even crushed seed tissue did not swell significantly when processed anhydrously. However, anhydrously processed specimens proved difficult to section. The difficulty was due to imperfect permeation of plastic into the seed tissues during embedding. An explanation of why anhydrously processed dry seed tissues are so difficult to embed in plastic is offered.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

A sensitive stain for aluminum in undecalcified cancellous bone

Aluminum is known to accumulate with age in bone and other tissues of humans, even in the absence of renal disease. Our study aimed to develop a histological staining method sufficiently sensitive to detect aluminum in plastic sections of undecalcified bone biopsies from healthy volunteers as well as from patients with renal and non-renal bone diseases. We used quantitative histomorphometry to measure the percentage of trabecular surface stained by aluminum and found that our new method was approximately 50% more sensitive for detecting aluminum than the Acid Solochrome Azurine (ASA) method which in turn was significantly more sensitive than the Aluminon method. Aluminon is widely used in pathology laboratories for diagnostic purposes despite concerns in the literature about Aluminon's limited sensitivity for aluminum. Our histomorphometric results showed that the newly developed method stained approximately 10% of the trabecular surface in bone sections from healthy controls, 38% from renal patients, 26% from patients with vitamin D deficiency, and 29% from patients with osteoporosis. Histomorphometric measurements of aluminum-stained trabecular surfaces in sections stained with ASA were consistent with those obtained in Walton-stained sections but proportionately lower. Moreover, the Walton and ASA methods stained aluminum at similar locations in adjacent bone sections. As the ASA and Walton methods are considerably more sensitive for bone aluminum than the Aluminon method, we recommend that either of them should be used in place of the Aluminon method for routine diagnostic purposes.     

A simplified paraffin embedding method for small botanical samples

Abstract

We present a simplified paraffin embedding method suitable for unsuberized or unlignified small botanical samples (diameter < 0.3 cm). Only 2 h are required to yield plant tissues embedded in paraffin for anatomical observation and molecular analysis. Our method achieved morphological preservation of cell structures and conservation of nucleic acids that were equivalent to the traditional protocol. Fourier transform infrared spectrometry showed that the degree of degradation of the cytoplasmic components (e.g., protein) resulting from our simplified protocol was similar to that of the traditional protocol. The DNA samples embedded using the simplified method was extractable and could be used for PCR analysis. The DNA quality was equivalent to that embedded using the traditional method.     

A New Antiroll Device for Cryostat Wax Sectioning

A new antiroll device has been developed to replace the antiroll guide plate for cryostat wax sectioning. With this device, a continuous ribbon of 3-4 μm sections can be obtained. The sections are flat, uncreased. and compression is reduced to a minimum.     

基于核局部线性嵌入的基因表达谱数据分类

针对局部线性嵌入算法（LocalLinearEmbedding,LLE）利用试凑法寻找近邻数耗时的缺陷性,提出一种增强的核局部线性嵌入算法（EnhancedKernelLocalLinearEmbedding,EKLLE）自动为样本分配邻域;该算法以高斯核函数为核心改进标准LLE距离度量准则,结合样本的类别信息,无需人工干预自动为样本设置不同的近邻数,克服了试凑法获得最优结果时需要大量时间;最后在各样本近邻数不相同的情况下对数据进行维数简约及待测样本分类。EKLLE算法有效地将高维基因表达谱数据映射到低维本质空间中,解决了传统LLE算法不能很好地处理合噪声或者稀疏数据的缺点。通过对比其他肿瘤样本分类实验,验证本文方法的实时性和精确性。     

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.     

Preparation of Thin Sections for Fourier Transform-Infrared Microscopy: a New Embedding Medium for Cellulosic Samples

Fourier Transform-Infrared [FT-IR] microscopy is a combination of instrumentation from which information can be derived about the structure and composition of materials; however, it presents unique problems for sample preparation. Traditional methods of preparing fiber cross sections employ embedding media such as methacrylates, epoxides and polyvinyl alcohols, all of which have groups in common with the cellulose molecule, and absorb in the same regions of the IR spectrum. Therefore, a new embedding method employing polystyrene has been developed for the preparation of cross and longitudinal sections of cellulosic fibers. Although polystyrene is a strong IR absorbing material, it can be completely removed from specimens prior to analysis. In addition, FT-IR spectra of cross sections have better resolution than conventional preparation methods employing ground samples prepared in a KBr disk.     

Telecentric design for digital-scanning-based HiLo optical sectioning endomicroscopy with an electrically tunable lens

Confocal endoscopy has been widely used to obtain fine optically sectioned images. However, confocal endomicroscopic images are formed by point-by-point scanning in both lateral and axial directions, which results in long image acquisition time. Here, an endomicroscope with telecentric configuration is presented to achieve nonmechanical and rapid axial scanning for volumetric fluorescence imaging. In our system, optical sectioning in wide-field fashion is obtained through HiLo imaging with a digital micromirror device. Axial scanning, without mechanical moving parts, is conducted by digital focus adjustment using an electrically tunable lens, offering constant magnification and contrast. We demonstrate imaging performance of our system with optically sectioned images using fluorescently labeled beads, as well as ex vivo mice cardiac tissue samples. Our system provides multiple advantages, in terms of improved scanning range, and reduced image acquisition time, which shows great potentials for three-dimensional biopsies of volumetric biological samples.     

Polar resin embedding for Macrotrachela quadricornifera

A new glycol methacrylate (GMA) polar resin technique was used for light microscopy and histochemistry of Macrotrachela quadricornifera. Animals were treated with cold aqueous solution of ethylene glycol (EG), then embedded in the cold. Animals required no conventional chemical fixation as EG stabilizes, dehydrates and cryopreserves their structure. In this way several enzymatic activities are preserved, along with morphology. Moreover, resin reticulate protects tissues against agressive reagents. As a consequence, it is possible to perform different staining procedures, in sequence, on the same section.     

Combined fluorescent antibody assay and viability staining for the assessment of the physiological states of Escherichia coli in seawaters

AIMS: A comparison of methods that combine the use of immune sera with specific fluorescent probes for testing viability at single cell level was performed in order to estimate different living attributes of Escherichia coli in natural seawater samples. METHODS AND RESULTS: Cell culturability was assayed by plate method, respiratory activity and membrane integrity were determined by an indirect fluorescent antibody assay, combined with 5-cyano-2, 3 ditolyl tetrazolium chloride and propidium iodide, respectively. Results showed the coexistence of different physiological states within the E. coli population, of which a large fraction (46%) of cells was actively respiring. CONCLUSIONS: The methodological approach used offer interesting perspectives in water pollution monitoring, particularly when the differentiation between dead and living E. coli cells is required for a more precise assessment of the bacteriological quality of seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggests the importance of knowledge of the viability status of faecal bacteria in aquatic environments as a fundamental issue for the preservation of public health; the availability of rapid analytical procedures for this purpose may find significant applications in the evaluation of the sanitary risk consequent to water use.