The application of lipid-soluble stains in plastic-embedded sections

Summary The present study was designed to develop a toutine method for direct demonstration and precize localisation of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4°C. Combination of lipid-soluble dyes with the periodic acid Schiff Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is casily applicable in routine histopathology practice.     

Unfixed material embedded in a methacrylate resin (Technovit 7100) for immunofluorescent staining

Wheat grains were embedded with or without fixation in Technovit 7100 or in paraffin. This enabled us to produce 4 microns sections for immunofluorescent staining to see whether serum of patients with baker's asthma contained IgG antibodies against wheat grains. Embedding without fixation in Technovit 7100 appeared to be suitable for immunofluorescent staining and gave superior results to protocols requiring fixation.     

Unfixed Material Embedded in A Methacrylate Resin (Technovit 7100) For Immunofluorescent Staining

Wheat grains were embedded with or without fixation in Technovit 7100 or in paraffin. This enabled us to produce 4 μm sections for imnwnofluorescent staining to see whether serum of patients with baker's asthma contained IgG antibodies against wheat grains. Embedding without fixation in Technovit 7100 appeared to be suitable for unmunofluorescent staining and gave superior results to protocols requiring fixation.     

Histochemical Demonstration of Enzyme Activities in Plastic and Paraffin Embedded Tissue Sections

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, β-galactosidase, and cytochrome oxidase in plastic embedded and routine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 μm, were far superior to frozen Sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.     

Improved Sensitivity for High Resolution in Situ Hybridization Using Resin Extraction of Methyl Methacrylate Embedded Material

An in situ hybridization procedure resulting in both high resolution and sensitivity was established by using the removable methyl methacrylate resin, Technovit 9100. Young bicel-lular pollen of tobacco (Nicotiana tabacum L. SR-1) was embedded in Technovit 9100 resin and sectioned. The resin was extracted with (2-methoxyethyl)-acetate followed by in situ hybridization with cRNA probes to detect cytoplasmic 18S/25S rRNA. Signal intensity obtained by this procedure was approximately twice as great as that obtained by an earlier procedure using Technovit 7100, a glycol methacrylate resin that cannot be removed from sections. This improvement in sensitivity made it possible to observe subcellular localization of small amounts of RNA as revealed by visualization of plastid 23S rRNA in a generative cell of Plumbago auriculata pollen.     

Effects of Storing Initiated 2-Hydroxyethyl Methacrylate Solutions for Embedding Tissues for Light Microscopy: Some Practical Implications

The effects of storing 2-hydroxy-ethyl methacrylate (HEMA) solutions for embedding tissues for light microscopy were studied using three commercially available HEMA embedding kits: Technovit 7100, Technovit 8100, and JB-4. These HEMA solutions were examined at various times of storage over a period of one year using a panel of physicochemical techniques including gas chromatography, tltration, viscosimetry, determination of the maximum polymerization temperature and the time required to reach the maximum temperature, and detection of degradation products of HEMA monomers by histochemical procedures. The quality of the resin blocks was examined by the observation of mini-folds in sections. Data obtained from these tests showed that the release of by-products as a result of the degradation of the HEMA monomer during storage of HEMA solutions does not occur. Development of cross-linking agents by transesterification of HEMA monomer was not detected either. Gradual decrease of the inhibitor concentration during storage proved to be the main cause of the reduction of shelf-life of HEMA solutions. Inconsistent tissue infiltration after storage may be due to decreased rates of tissue penetration as a result of HEMA chain lengthening. Guidelines for safe and economical handling of HEMA mixtures are given.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo.

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F1 hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat x maize hybrid embryos.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Glycol Methacrylate Embedding of Algmate-Polylysine Microencapsulated Pancreatic Islets

A method for processing and embedding alginate-polylysine microencapsulated pancreatic tissue in glycol methacrylate resin (GMA) is described. Fixation in 4% phosphate buffered formaldehyde, processing in ascending concentrations of glycol methacrylate monomer and embedding in Technovit 7100 results in well preserved morphological details of hydrogels, hydrogel-cell interfaces, and encapsulated pancreatic tissue. Routine staining with Loeffler's methylene blue, hematoxylin and eosin, and Romanovsky-Giemsa gave excellent images of the GMA embedded alginate polylysine membrane and tissues allowing cells on the outside of the capsule to be analyzed effectively as part of the foreign body reaction against the capsule membrane.     

Glycol Methacrylate Embedding of Algmate-Polylysine Microencapsulated Pancreatic Islets

A method for processing and embedding alginate-polylysine microencapsulated pancreatic tissue in glycol methacrylate resin (GMA) is described. Fixation in 4% phosphate buffered formaldehyde, processing in ascending concentrations of glycol methacrylate monomer and embedding in Technovit 7100 results in well preserved morphological details of hydrogels, hydrogel-cell interfaces, and encapsulated pancreatic tissue. Routine staining with Loeffler's methylene blue, hematoxylin and eosin, and Romanovsky-Giemsa gave excellent images of the GMA embedded alginate polylysine membrane and tissues allowing cells on the outside of the capsule to be analyzed effectively as part of the foreign body reaction against the capsule membrane.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

A novel thin section preparation and staining protocol to increase contrast and resolution of cell details for light microscopy

ABSTRACT

We developed a novel sectioning and staining method to make high contrast, high resolution sections of plant tissue for light microscopy. Specimens of teosinte (Zea mays L., ssp. mexicana) root tips were fixed and embedded in Technovit 7100? plastic resin. Thin sections, 1?2.5 μm, were cut and mounted on glass slides. The sections were either treated with RNase or not, then stained with 0.1% toluidine blue O and observed through ∞/0 objective lenses. For light microscopy, the enzyme staining procedure increased resolution and contrast. High magnification ∞/0 objective lenses produced high quality images for digital photography without using a coverslip or immersion oil. Our slide preparation and microscopic analysis were less labor intensive and more rapid than previous methods and enabled rapid and precise alignment of serial transverse sections for both tracking cell lineages and tissue measurements.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Optimized preservation of CNS morphology for the identification of glycogen in the Pompe mouse model.

Pompe disease (glycogenosis type II) is a rare lysosomal disorder caused by a mutational deficiency of acid alpha-glucosidase (GAA). This deficiency leads to glycogen accumulation in multiple tissues: heart, skeletal muscles, and the central nervous system. A knockout mouse model mimicking the human condition has been used for histological evaluation. Currently, the best method for preserving glycogen in Pompe samples uses epon-araldite resin. Although the preservation by this method is excellent, the size of the tissue is limited to 1 mm(3). To accurately evaluate brain pathology in the Pompe mouse model, a modified glycol methacrylate (JB-4 Plus) method was developed. This approach allowed the production of larger tissue sections encompassing an entire mouse hemisphere (8 x 15 mm) while also providing a high level of morphological detail and preservation of glycogen. Application of the JB-4 Plus method is appropriate when a high level of cellular detail is desired. A modified paraffin method was also developed for use when rapid processing of multiple samples is a priority. Traditional paraffin processing results in glycogen loss. The modified paraffin method with periodic acid postfixation resulted in improved tissue morphology and glycogen preservation. Both techniques provide accurate anatomic evaluation of the glycogen distribution in Pompe mouse brain.     

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.     

Distribution of [14C] Dichlorophenoxyacetic Acid in Cultured Zygotic Embryos of Zea mays L.

The uptake of 2,4-dichlorophenoxyacetic acid (2,4-D), necessary for the in vitro induction of callus formation and somatic embryogenesis in cultured immature maize embryos, was quantified after culture on nutrient medium with [¹⁴C]2,4-D. The identity of the ¹⁴C label in the embryos was determined by high performance liquid chromatography (HPLC), and its distribution within embryos was visualized on sections of plastic embedded material. Quantification of the ¹⁴C label after a pulse label of 16 h showed a hundredfold accumulation of 2,4-D in the embryos with respect to the initial medium concentration. During tissue processing for in situ detection of ¹⁴C, however, up to 70% of the label disappeared because of the embedding process. The best structural preservation was obtained after ethanol-mediated infiltration of Technovit 7100. Water-mediated infiltration of Technovit 7100 gave the highest retention of ¹⁴C. HPLC analysis showed that more than 95% of the residual ¹⁴C label found in embryos was still 2,4-D. Autoradiography showed that the embryogenic inbred line A188 contained ¹⁴C label in distinct regions of the scutellum, coleoptile, and suspensor. The nonembryogenic inbred line A632 contained more label after 16 h of culture in a different distribution compared with A188. Subculture of the embryos for 24 and 72 h and histologic analysis showed that cell proliferation and callus formation were restricted to specific regions of the embryo in both inbred lines. The pattern of 2,4-D distribution did not codistribute with regions of proliferation, indicating that 2,4-D is not the only trigger for proliferation. Received August 18, 1997; accepted February 12, 1998     

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 trial of histological preparation of early mouse embryos with the Technovit 7100 system

A reproducible procedure was devised for making histological preparations of early mouse embryos cultured in vitro. This method was characterized by use of a resinous embedding material, Technovit 7100, and a 96-well U-bottomed microplate, which enabled efficient rinsing and dehydration of fixed embryos without loss. Technovit 7100 polymerized or hardened at room temperature slowly enough to allow sedimentation of embryos to the bottom, which enabled more correct prediction of the place of the embryos at sectioning. The present method will lead to easier microscopic observation of early embryos in embryology.     

A New Preparation Method For Several Histopathological Examinations On A Single Block

A new method of specimen preparation is described permitting several studies such as routine staining, histochemistry, enzyme histochemistry, immunohistochemistry, and electron microscopy on a single block of biopsy specimens. Tissues are immersed in the fixative, which primarily stabilizes carbohydrate moieties, and embedded in the mixture of JB-4, methylmethacrylate and divinylbenzcne. The resin is polymerized at 4 C Thin sections (1-2 microns) are obtained with a sliding microtome, and ultrathin sections (60-90 millimicrons) with a ultramicrotome. The sections are stained directly with various conventional procedures without removing the embedding resin. This preparation method offers a potentially useful tool for histopathological studies on biopsy specimens.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.