Embedding and Sectioning Undecalcified Bone, and its Application to Radioautography

A method is described for embedding and sectioning hard, undecalcified bone, which is designed for use by technical personnel. Bone fixed in a variety of ways is progressed through alcohols to ether-alcohol and then infiltrated with ether-alcohol solvented plastic (plasticized nitrocellulose) by a combination of centrifugation and high pressure embedding technics. The ether-alcohol is evaporated in a partially closed container in a manner similar to that employed in celloidin embedding, but differs from the latter by the removal of all of the solvent. Celloidin is the source of nitrocellulose and Amoil-S, the added plasticizer. Undecalcified adult bone of all types is readily cut at a thickness of 5-8μ on a heavy duty sliding microtome. The sections are then mounted on gelatinized slides. The procedures for preparing strip film radio-autograms of bone sections and subsequent staining of the preparation are given. The results obtained are illustrated.     

A Simple Screening Procedure for Evaluating Central Nervous System Tissue Sections Showing Structural and Cytochemical Alterations of the Blood-Brain Barrier

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 μm and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.     

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.     

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.     

Piccolyte investments for better section cutting.

Piccolyte 115 (beta-pinence polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Freeze drying and freeze substitution combined with low temperature-embedding

Summary An X-ray microanalytical and morphological investigation was carried out on rapidly frozen freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze substitution and freeze drying. The investigation also included an analysis of specimens which had been infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl-HM20. The method of freeze drying, followed by embedding and polymerisation at low temperatures in vacuo was found to give satisfactory results, comparable with more tedious and hazardous freeze substitution technique.     

Improvement of Automatic In-Gel Digestion by in Situ Alkylation of Proteins

We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (1D) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with “Mascot” search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli 1D SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; –S–CH₂CONH₂) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM; –S–CH₂CH₂CONH₂) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.     

The use of human fibroblasts grown on microcarriers for the formation of the connective tissue equivalent

Live equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the live tissue equivalent amounts to three--four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Continuous perfusion of mammalian cells embedded in agarose gel threads

A method for perfusing cells by embedding them in fine agarose gel threads is described and characterized. The rate of diffusion of a metabolite into the gel threads is determined by 31P-NMR spectroscopy. This perfusion method is shown to enable Chinese hamster lung fibroblasts (CHLF) to remain in a metabolically active state with high levels of intracellular ATP for many hours.     

The application of polyethylene glycol (PEG) to electron microscopy

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.     

Use of Human Fibroblasts Grown on Microcarriers for Formation of Connective Tissue Equivalent

Living equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the living tissue equivalent amounts to three–four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

A Rapid Rosin-Celloidin-Paraffin method for Embedding Tissues

Pieces of tissue of various sizes or tissue fragments are dehydrated in 95% alcohol, cleared, washed with ether and infiltrated with a solution containing parloidin 9.6 g., camphor 3.0 g., absolute alcohol 200.0 ml., ether 200.0 ml., rosin 45.0 g. and castor oil 10 drops. After evaporation to the desired consistency, the mass is hardened with chloroform vapor, trimmed, passed through 3 changes of xylene to remove the rosin, embedded in paraffin, sectioned serially, stretched and stained as for paraffin methods. Methods of defatting tissues and detailed procedure for embedding fragments of bone marrow by this method are given.     

Collagen-gel-embedded three-dimensional culture of human thyroid epithelial cells: comparison between the floating sandwich method and the dispersed embedding method.

Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells.     

Histology of plastic embedded amphibian embryos and larvae

Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development, and regeneration. For the analysis of embryos and larva with altered gene expression in gain-of-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwave-assisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple, and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos.     

A Quick Preparative Method for Electron Microscopy Observations of Delicate Objects Using Alginate Embedding Medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

A quick preparative method for electron microscopy observations of delicate objects using alginate embedding medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

Classification of microarrays to nearest centroids

MOTIVATION: Classification of biological samples by microarrays is a topic of much interest. A number of methods have been proposed and successfully applied to this problem. It has recently been shown that classification by nearest centroids provides an accurate predictor that may outperform much more complicated methods. The 'Prediction Analysis of Microarrays' (PAM) approach is one such example, which the authors strongly motivate by its simplicity and interpretability. In this spirit, I seek to assess the performance of classifiers simpler than even PAM. RESULTS: I surprisingly show that the modified t-statistics and shrunken centroids employed by PAM tend to increase misclassification error when compared with their simpler counterparts. Based on these observations, I propose a classification method called 'Classification to Nearest Centroids' (ClaNC). ClaNC ranks genes by standard t-statistics, does not shrink centroids and uses a class-specific gene-selection procedure. Because of these modifications, ClaNC is arguably simpler and easier to interpret than PAM, and it can be viewed as a traditional nearest centroid classifier that uses specially selected genes. I demonstrate that ClaNC error rates tend to be significantly less than those for PAM, for a given number of active genes. AVAILABILITY: Point-and-click software is freely available at http://students.washington.edu/adabney/clanc.     

Secretion of peptidylglycine alpha-amidating monooxygenase (PAM) from rat salivary glands.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a regulating enzyme to synthesize the biologically active hormones having carboxy-terminal amide. In the present study we investigated secretion of the enzyme from rat saliva. Property of PAM in the saliva was similar to that in the submandibular gland. Both enzymes showed similar pH optimum at 5.0 and optimal ascorbic acid concentration at 2.5 mM. But molecular size of PAM in the saliva was 75 kDa in the gel permeation chromatography on Superose 12 column, while the size in the submandibular gland was 25 kDa. After the treatment with trypsin, PAM in the saliva was converted to a small size molecule, which is similar to the size in rat submandibular gland. These and other data indicate that a native molecular size of PAM is secreted into saliva and plays some physiological roles.