Glycol Methacrylate Embedding Research Articles

Plastic embedding for precise imaging of large-scale biological tissues labeled with multiple fluorescent dyes and proteins.

Resin embedding of multi-color labeled whole organs is the primary step to preserve structural information for visualization of fine structures in three dimensions. It is essential to study the morphological characteristics, spatial and positional relationships of the millions of neurons, and the intricate network of blood vessels with fluorescent labels in the brain. However, the current resin embedding method is inadequate because of incompatibilities with fluorescent dyes, making it difficult to reconstruct a variety of structures for the interpretation of their complex spatial relationships. We modified the resin embedding method for large biological tissues labeled with multiple fluorescent dyes and proteins through different labeling strategies. With TrueBlack as the background fluorescence inhibitor in the glycol methacrylate (GMA) embedding, we referred to the method as GMA-T (Glycol methacrylate with TB). In the GMA-T embedded mouse brains, structures labeled with fluorescent proteins and dyes were visualized in millimeter-scale networks with sub-cellular resolution, allowing quantitative analysis of different anatomical structures in the same brain, including neurons and blood vessels. In combination with high-resolution whole-brain imaging, it is possible to obtain a variety of fluorescence labeled structures in just a few days. We quantified the distribution and morphology of the tdTomato-labeled vasoactive intestinal polypeptide (VIP) neurons and the BSA-FITC labeled blood vessels in the same brain. These results demonstrated that VIP neurons and blood vessels have their own unique distribution patterns and morphological characteristics among cortical regions and different layers in cerebral cortex, and there was no significant correlation between VIP neurons and vessels. This approach provides a novel approach to study the interaction among different anatomical structures within large-volume biological samples labeled with multiple fluorescent dyes and proteins, which helps elucidating the complex anatomical characteristics of biological organs.

Into the depths: Techniques for in vitro three-dimensional microtissue visualization.

Three-dimensional (3-D) in vitro platforms have been shown to closely recapitulate human physiology when compared with conventional two-dimensional (2-D) in vitro or in vivo animal model systems. This confers a substantial advantage in evaluating disease mechanisms, pharmaceutical drug discovery, and toxicity testing. Despite the benefits of 3-D cell culture, limitations in visualization and imaging of 3-D microtissues present significant challenges. Here we optimized histology and microscopy techniques to overcome the constraints of 3-D imaging. For morphological assessment of 3-D microtissues of several cell types, different time points, and different sizes, a two-step glycol methacrylate embedding protocol for evaluating 3-D microtissues produced using agarose hydrogels improved resolution of nuclear and cellular histopathology characteristic of cell death and proliferation. Additional immunohistochemistry, immunofluorescence, and in situ immunostaining techniques were successfully adapted to these microtissues and enhanced by optical clearing. Utilizing the Clear(T2) protocol greatly increased fluorescence signal intensity, imaging depth, and clarity, allowing for more complete confocal fluorescence microscopy imaging of these 3-D microtissues compared with uncleared samples. The refined techniques presented here address the key challenges associated with 3-D imaging, providing new and alternative methods in evaluating disease pathogenesis, delineating toxicity pathways, and enhancing the versatility of 3-D in vitro testing systems in pharmacological and toxicological applications.

Glycol methacrylate embedding for the histochemical study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum.

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract (GIT) has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy’s solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs compared with controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail.

Staining histological lung sections with Sudan Black B or Sudan III for automated identification of alveolar epithelial type II cells

Alveolar epithelial type II (AE2) cells produce, store and secrete pulmonary surfactant and serve as progenitor cells for the alveolar epithelium. They are thus an interesting target in wide fields of pulmonary research. Stereological methods allow their quantification based on measurements on histological sections. A proper AE2 cell quantification, however, requires a method of tissue processing that results in little tissue shrinkage during processing. It was recently shown that a primary fixation with a mixture of glutaraldehyde and formaldehyde, postfixation with osmium tetroxide and uranyl acetate and embedding in glycol methacrylate fulfills this requirement. However, a proper quantification, furthermore, requires a secure identification of the cells under the microscope. Classical approaches using routine stainings, high magnifications and systematic uniform random sampling can result in a tedious counting procedure. In this article we show that Sudan Black B and Sudan III staining in combination with the previously described “low shrinkage method” of tissue processing result in good staining of lamellar bodies of AE2 cells (their storing organelles of surfactant) and thus provide a good signal of AE2 cells, which allows their easy and secure identification even at rather low magnifications. We further show that this signal enables automated detection of AE2 cells by image analysis, which should make this method a suitable staining method for the recently developed and more efficient proportionator sampling.

Glycol methacrylate: the art of embedding and serial sectioning

Glycol methacrylate (GMA) is a popular embedding medium used for high resolution light microscopy. Since the introduction of the GMA embedding method by Feder and O’Brien (1968, Am. J. Bot. 55: 123–142), improvements have been made to this technique. The purpose of our work is to detail some important advancements in the GMA embedding method and discuss different approaches to ensure successful processing and serial sectioning of GMA blocks. The best sectioning method is to combine the use of Ralph glass knives with a rotary microtome with a retraction return stroke. Moreover, softening the polymerized blocks with the addition of polyethylene glycol 400 during polymerization allows the blocks to be sectioned using a conventional rotary microtome with a disposable knife system.

Morphometric examination of the equine adult and foal lung.

To understand the mechanisms of airway inflammation associated with equine diseases such as Rhodococcus equi infection, we must identify baseline "normal" structural characteristics of the horse lung. To develop a detailed understanding of the morphology of the horse lung, we adapted and applied stereological methods to the lungs from healthy adult horses (N = 4) and 1-day (N = 5) and 30-day (N = 5) old foals. The left lung was fixed in situ by intrabronchial instillation of glutaraldehyde/paraformaldehyde fixative at 25 cm H2 O column and sampled using a fractionator design followed by embedding in glycol methacrylate. The lung was characterized into parenchyma and non-parenchyma, where median parenchymal density was 81.0% in 1-day-old foals, 84.4% in 30-day-old foals and 93.7% in adult lungs. The median volume density of alveolar airspace per lung was 45.9% in 1-day-old, 55.5% in 30-day and 66.9% in adult horse lungs. The median alveolar surface area increased with age, from 205.3 m(2) , 258.2 m(2) , and 629.9 m(2) in 1-day-old foals, 30-day-old foals, and adults, respectively. While the median alveolar surface density decreased with age, the mean linear intercept (mean free distance within acinar airspaces) increased with age. Alveolar surface area was greater than endothelial surface area within each lung. The ratio between alveolar and endothelial surface density remains unchanged with age. The median endothelium surface area was 106.2 m(2) in 1-day, 147.5 m(2) in 30-day, and 430 m(2) in adult lungs. The data suggest the foal lung is functionally developed and postnatal lung development and remodelling is driven by alveolar expansion paralleled with angiogenesis.

Alterations of mouse lung tissue dimensions during processing for morphometry: A comparison of methods

Preservation of original tissue dimensions is an essential prerequisite for morphometric studies. Shrinkage occurring during tissue processing for histology may severely influence the appearance of structures seen under the microscope and stereological calculations. Therefore, shrinkage has to be avoided so that estimates obtained by application of unbiased stereology are indeed unbiased. The present study investigates the alterations of tissue dimensions of mouse lung samples during processing for histology. Different fixatives as well as embedding protocols are considered. Mouse lungs were fixed by instillation of either 4% formalin or a mixture of 1.5% glutaraldehyde/1.5% formaldehyde. Tissue blocks were sampled according to principles of stereology for embedding in paraffin, glycol methacrylate without treatment with osmium tetroxide and uranyl acetate, and glycol methacrylate including treatment with osmium tetroxide and uranyl acetate before dehydration. Shrinkage was investigated by stereological measurements of dimensional changes of tissue cut faces. Results show a shrinkage of the cut face areas of roughly 40% per lung during paraffin embedding, 30% during "simple" glycol methacrylate embedding, and <3% during osmium tetroxide/uranyl acetate/glycol methacrylate embedding. Furthermore, the superiority of the glutaraldehyde-containing fixative regarding shrinkage is demonstrated. In conclusion, the use of a glutaraldehyde-containing fixative and embedding in glycol methacrylate with previous treatment of the samples with osmium tetroxide and uranyl acetate before dehydration is recommended for stereological studies of the mouse lung.

The use of NaOCl in combination with CHX produces cytotoxic product

The combination of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) yields a "precipitate potentially toxic" (PPT). The aim of this study was to evaluate the tissue response to implanted polyethylene tubes filled with PPT-soaked fibrin sponge. Forty rats received four polyethylene tubes each; each tube was filled with fibrin sponge soaked by 2.5 % NaOCl, 2.0 % CHX, PPT (2.5 % NaOCl plus 2.0 % CHX), or not soaked (control). The observation time points were 7, 15, 30, 60, and 90 days. At each time point, eight animals were killed, and the tubes and surrounding tissues were removed, fixed, and prepared for light microscopic analysis by performing glycol methacrylate embedding, serial cutting into 3-μm sections, and hematoxylin-eosin staining. Qualitative and quantitative evaluations of the reactions were performed. Results were statistically analyzed by Kruskal-Wallis test (p < 0.05). All chemical solutions caused moderate reactions at 7 days. On day 30, PPT group was more cytotoxic than the control group and the CHX group (p < 0.05). On days 15 and 60, PPT group was more cytotoxic than the control group (p < 0.05). On day 90, there was no statistically significant difference between the different groups. PPT is more cytotoxic than NaOCl and CHX alone, particularly in the short term. Protocols which suggest the use of CHX and NaOCl must be revised because this mixture produces cytotoxic product.

Large plant samples: how to process for GMA embedding?

It is often necessary to process large plant samples for light microscopy studies, but due to structural characteristics of plant tissues, especially intercellular spaces, large vacuoles, and phenolic substances, results are often unsatisfactory. When large samples are embedded in glycol methacrylate (GMA), their core may not polymerize, remaining soft and moist and making it difficult to cut microtome sections. This situation has been erroneously interpreted as the result of poor infiltration, when the soft core of these samples is actually the result of incomplete polymerization. While GMA is in fact present inside samples, unsatisfactory polymerization results from rapid external polymerization that does not allow sufficient hardener to reach the sample core, while the relatively large volume of GMA inside the tissue block also dilutes the hardener. In this chapter we propose a new method for processing large plant specimens that avoids these problems by: (1) slowing the polymerization process through cooling in order to permit the penetration of hardener into the sample core and (2) increasing the hardener:GMA ratio to aid polymerization of the sample core.

Tissue Reaction to Silver Nanoparticles Dispersion as an Alternative Irrigating Solution

Introduction Nanomaterials have been used to create new consumer products as well as applications for life sciences and biotechnology. The aim of this study was to evaluate the tissue response to implanted polyethylene tubes filled with fibrin sponge embedded with silver nanoparticles dispersion. Methods Thirty rats received individually 4 polyethylene tubes filled with sponge embedded in 47 ppm, 23 ppm silver nanoparticles dispersion, 2.5% sodium hypochlorite, or with no embedding as control. The observation periods were 7, 15, 30, 60, and 90 days. After each period of time, 6 animals were killed, and the tubes and surrounding tissue were removed, fixed, and prepared to be analyzed in light microscope with glycol methacrylate embedding, 3-μm serial cutting, and hematoxylin-eosin stain. Qualitative and quantitative evaluations of the reactions were performed. Results Both materials caused moderate reactions at 7 days. The response was similar to the control on the 15th day with 23 ppm silver nanoparticles dispersion and 2.5% sodium hypochlorite and on the 30th day with 47 ppm silver nanoparticles dispersion. Conclusions It was possible to conclude that silver nanoparticles dispersion was biocompatible especially in a lower concentration.

Methodological Issues Related to Pathological Evaluation of Coronary Artery Stents

Intravascular placement of metallic stents is a common treatment of stenosed coronary arteries; however, they are associated with risk of in-stent restenosis and late-stent thrombosis. Pathological evaluation of stented coronary arteries is needed to characterize stent-tissue interactions to improve the safety and efficacy of coronary stents. Thus, high-quality tissue sections are required with excellent preservation of the morphology of the stented artery, particularly at the stent-tissue interface. In this study, formalin-fixed and dehydrated stented coronary artery segments from cases of cardiac transplantation and routine autopsies were used. In a vacuum chamber, the specimens were immersed in infiltration solution (benzoylperoxide with hydroxyethyl methacrylate) from the JB-4 Plus Embedding Kit® (Polysciences, Inc.). The specimens were then transferred into the embedding medium (infiltration solution with polyethylene glycol) and allowed to polymerize under anaerobic conditions. Next, the specimens were cut into 5-mm-thick blocks with a rotary saw, sectioned with a JB-4 microtome, and stained with hematoxylin and eosin and Masson's trichrome. Morphology of all coronary artery segments, including the stent-tissue interface, was readily visible; however, to achieve adequate staining intensity, prolonged hematoxylin staining was required. Some stents were displaced from their original position during sectioning and staining. Attempts to use another plastic embedding medium (Technovit 9100 methyl methacrylate) have been unsuccessful so far. This was primarily related to failure of the block to adhere to the holder during sectioning. In conclusion, with the use of glycol methacrylate embedding technique the morphology of the stent-tissue interface of stented arteries can be readily observed by microscopy. Failure to maintain the stent in its original position is an important limitation, possibly related to the relative softness of the medium. Refining the methodology may lead to a more efficient protocol, enabling more specialized staining as well as the use of harder plastic media such as methyl methacrylate. (The J Histotechnol 32(4):151–154, 2009)

Immunohistochemical Detection of Ki67 in Glycol Methacrylate Sections Using Unfixed Tissues and Alkaline Antigen Retrieval Techniques

Assessing cell proliferation through immunohistochemical procedures is an important component of many research projects. Unfortunately, published protocols often yield inconsistent results in individual laboratories. We present a method for glycol methacrylate embedding and antigen retrieval with alkaline solutions to detect Ki67 antigen. In brief, fresh tissues are dehydrated through a series of catalyzed resin solutions, and then embedded for standard microtomy at 1.5–2 μm. Cut sections were mounted on slides and pretreated with a 0.2% NaOH solution for 1.5–2 minutes and then stained to detect Ki67 according to standard immunohistochemical protocols. Secondary detection reagents tested in the development of this protocol included HRP/DAB combinations, Au/Ag staining procedures, and fluorescent secondary antibodies. Antigen sites could not be demonstrated in aldehyde-fixed tissues, but were readily demonstrated in the unfixed tissues after alkaline antigen retrieval. The method described is a simple, inexpensive, and rapid procedure to allow antigen detection. The method may have additional utility in the demonstration of other nuclear antigens. (The J Histotechnol 32(2):60 – 63, 2009)Submitted January 18, 2009; accepted with revisions March 25, 2009

Immunohistochemical Detection of Ki67 in Glycol Methacrylate Sections Using Unfixed Tissues and Alkaline Antigen Retrieval Techniques

Assessing cell proliferation through immunohistochemical procedures is an important component of many research projects. Unfortunately, published protocols often yield inconsistent results in individual laboratories. We present a method for glycol methacrylate embedding and antigen retrieval with alkaline solutions to detect Ki67 antigen. In brief, fresh tissues are dehydrated through a series of catalyzed resin solutions, and then embedded for standard microtomy at 1.5–2 μm. Cut sections were mounted on slides and pretreated with a 0.2% NaOH solution for 1.5–2 minutes and then stained to detect Ki67 according to standard immunohistochemical protocols. Secondary detection reagents tested in the development of this protocol included HRP/DAB combinations, Au/Ag staining procedures, and fluorescent secondary antibodies. Antigen sites could not be demonstrated in aldehyde-fixed tissues, but were readily demonstrated in the unfixed tissues after alkaline antigen retrieval. The method described is a simple, inexpensive, and rapid procedure to allow antigen detection. The method may have additional utility in the demonstration of other nuclear antigens. (The J Histotechnol 32(2):60 – 63, 2009)Submitted January 18, 2009; accepted with revisions March 25, 2009

Effect of histological processing and methacrylate sectioning on the area of gill tissue in teleost

Deformation of biological tissues may occur during histological processing and results in loss of accuracy when quantitative information about cells, tissues and organs is necessary. In this study, the gill tissue from armored catfish (Pterygoplichthys anisitsi) was quantified in each step of processing using the stereological principles. During processing for glycol methacrylate embedding, gill tissue from shrinks significantly but regains its original dimensions after sectioning.

A method for the histochemical localization of digestive enzymes in whole freeze-substituted larval fish embedded in glycol methacrylate

In order to study the localization of digestive enzymes in larval walleye (Sander vitreus vitreus), a novel method of low-temperature processing of whole, unfixed larvae and subsequent embedding in glycol methacrylate (GMA) was developed. Larval walleye aged 2–10 days post hatch were flash-frozen in liquid nitrogen and transferred into pre-chilled acetone for 12 h at a temperature of −25 °C. Acetone was gradually replaced with increasing concentrations of GMA resin monomer over a 6-h period. The polymer (100%) was further allowed to infiltrate the larvae for 36 h. Resin-embedded larvae were transferred to embedding moulds and polymerized overnight at −25 °C. Four micrometre sections were stained to identify either alkaline phosphatase, non-specific esterase, aminopeptidase M or dipeptidyl peptidase IV. All enzymes studied were readily detected and accurately localized, and no enzyme diffusion was observed. Therefore, freeze substitution combined with low-temperature GMA embedding allows the maintenance of excellent tissue morphology and accurate enzyme localization in whole larval walleye specimens from 2 to 10 days post hatch. It is recommended, however, that samples be frozen in pre-cooled fluorocarbon-based liquid coolants in order to assure optimal tissue preservation.

Histological approaches for high-quality imaging of zooplanktonic organisms

The investigation of the internal organization of zooplankton communities provides important information on the plankton biology with special interest for the study of ecological processes. Zooplanktoners can play a structural function as indicators for ecosystem health or stress, but their study using histological techniques is still limited. Here we report that the internal structure of zooplanktonic organisms can be facilely observed by a histological approach that combines optimal fixation and processing with a plastic resin (glycol methacrylate) embedding, resulting in increased tissue resolution. Using copepods, organisms that can dominate zooplankton assemblages, as models, collected from a tropical ecosystem (Paraibuna river, Brazil), we showed fine histological details of their muscular, nervous and digestive systems, structure of appendages and cell features. Critical advantages of this approach are that it permits optimal preservation and adequate handling of the organisms (embedded in agar after fixation) for further histological processing and investigation. This is important because it prevents both mechanically induced artifacts and loss of these diminutive organisms during the different steps of processing. Moreover, embedding in plastic resin showed a superior imaging of copepod internal structures compared to paraffin embedding. The use of glycol methacrylate is advantageous over paraffin/paraplast embedding by avoiding heat damage, tissue retraction and allowing faster embedding procedure and better tissue resolution. The value of histological approaches in enabling high-quality imaging of the internal structure of copepods is particularly important because these organisms can be used as indicators of environmental changes.

High-Resolution Immunohistochemistry on Improved Glycol Methacrylate—Resin Sections

Glycol methacrylate (GMA) embedding resins can be im­proved by the use of self-prepared mixtures. By the use of 99% 2-hydroxyethyl-methacrylate (i.e., GMA), the dibenzoyl peroxide content of solution A can be reduced and in addition the proportion of accelerator solution that is necessary for polymerization. The resulting lower polymerization temperature is advantageous in maintaining the tissue structure and also antigenic activity. Most immunohistochemistry (IHC) reactions can be made successfully without retrieval treatment if a suitable tissue fixative has been used. The required high sensitivity needed for IHC on GMA-resin embedded tissue sections can be achieved with SuperPic-Ture™ horseradish peroxidase polymer conjugates (Zymed) and visualised with antihorseradish peroxidase fluorochrome conjugates or Movat silver-intensified DAB or immunogold-silver staining. Outstandingly clean and clear results can be obtained by using phosphate-buffered saline/Tris buffer mixtures with 0.1% Extran for rinsing. In this work, we detail instructions for the 99% GMA resin embedding and step-by-step procedures for IHC on GMA-resin sections for fluorescence and bright-field microscopy

Glycolmethacrylate Is Superior to Methylmethacrylate for Histologic Evaluation of Biodegradable Polymer Scaffolds Used for Vascular Tissue Engineering

The use of autologous arteries or veins is the “gold standard” for cardiovascular bypass grafting to repair congenital defects or diseased vessels. Nonetheless, a limited supply of healthy vessels for harvest necessitates the use of alternative graft materials. Biodegradable, tissue-engineering scaffolds offer much promise as a grafting material. However, to fully evaluate the efficacy of these scaffolds, multiple analyses must be performed, including histological evaluation. Routine processing and embedding in paraffin may not adequately infiltrate and support biodegradable scaffolds; therefore, alternate processing methods, such as plastic embedding, must be considered to produce high-quality histological sections. Here, we compare two methods for embedding scaffolds in plastic, using either glycolmethacrylate or methylmethacrylate. Overall, we found that glycolmethacrylate embedding and associated processing techniques were clearly superior to methylmethacrylate procedures as illustrated by intact sections with nicely preserved cells. (The J Histotechnol 29:245, 2006)Submitted October 1, 2006; accepted with revisions October 30. 2006

Histological Techniques for Porous, Absorbable, Polymeric Scaffolds, Used in Tissue Engineering

Tissue engineering is the development of biologically based materials for tissue replacement, often by combining cells with an absorbable, porous scaffold material. Absorbable materials are sensitive to histological processes, and the fragile nature of tissue-engineered devices compounds these problems. This study compared different methods of embedding and sectioning tissue-engineered constructs, including paraffin, cryostat, and hard tissue techniques. It was found that glycol methacrylate embedding is more time consuming and expensive but retains the structure and results in minimal background staining as compared with other techniques. It was found that cryosectioning using an adapted tape transfer technique is inexpensive and quick, but at this time has limited staining options due to high background staining. It will be of future interest to modify staining protocols to further this technique. (The J Histotechnol 26:57, 2003)Submitted August 28, 2002; Accepted with revisions January 13, 2003

Optimization of glycol methacrylate embedding of large specimens in neurological research. Study of rat skull–brain specimens after implantation of polyester meshes

Advances in neuroscience require better anatomical knowledge of neuronal architecture and structural details. Optimal embedding techniques are the basis for precise morphometric studies in section series as well as for the evaluation of tissue specimens or implants of differing hardness. There are very few methods for preparing large specimens by resin embedding, although resins such as polyethylene glycol (PEG) and methyl methacrylate (MMA) are presently in use. However, these methods have proven to be laborious and sometimes unsatisfactory for serial sectioning. While glycol methacrylate embedding (GMA) is suitable for smaller specimens, it results in inadequate infiltration and polymerization in blocks larger than 1×1×0.2 cm. We present an improved technique using GMA, which permits both standardized embedding of 4×2×2 cm blocks and preparation of section series. This method was developed for preserving skull-brain specimens from rats with polyester-mesh implants. The excellent preservation of cellular details allowed the assessment of local tissue reaction to foreign-body material in situ. Advantages of this method are: (1) No toxic catalysts or solvents are used (as opposed to MMA and current GMA processes); (2) Laborious routines in stretching and mounting of sections are not necessary (in contrast to PEG and MMA); (3) No deplastination is required before staining (in contrast to PEG and MMA); (4) Excellent morphologic preservation of various tissue is achieved.