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PubMed1998

Preservation and staining of myelinated nerve fibers.

Six procedures are given for preservation of myelinated nerve fibers for light or electron microscopic studies. These procedures fall into two main categories: those with and those without aldehyde fixation. Essentially different effects are attained by application of tannic acid, saline, microwave or conventional heating, or a decreased temperature. All procedures end in osmication. Three main aspects of myelinated fiber morphology are taken into account when judging the quality of their preservation: axon, myelin sheath, and axon/myelin coherence. Each aspect can be preserved excellently, but always in combination with a less superior quality of the other two aspects. Superior myelin quality is attained using microwave irradiation, either with aldehydes to which tannic acid is added or without aldehyde fixatives. Superior axon quality is attained with aldehydes and (conventional) heating. Axon/myelin coherence is best preserved by decreasing the temperature during the rinse with saline. Another two procedures provide good, though less superior, preservation of both axon and axon/myelin coherence. Next, the fixed tissue is embedded in plastic blocks from which semithin and ultrathin plastic sections are cut for light and electron microscopy, respectively. In addition to the standard procedure for toluidine blue staining on semithin sections, two microwave-supported procedures are described, which can be used as alternatives if the staining result is unsatisfactory. Furthermore, a toluidine blue staining procedure is described for glycol methacrylate (GMA)-embedded material, which can be used if larger sections are needed.

Aldehydes↗
PubMed2004

A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques.

The purpose of this study was to compare the level of immunogold labelling of both osmicated and non-osmicated epoxy sections when subjected to different antigen retrieval, etching and incubation temperature for the antibodies. Pure IgG protein gels were produced by glutaraldehyde fixation, eventually postfixed with 1% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were antigen retrieved in citrate solution at 95 or 144 degrees C and eventually etched with NaIO4. Immunogold labelling with anti-IgG was performed at 4 degrees C overnight or at 60 degrees C for 1 h. The level of labelling for osmicated gels was 140% higher when heated at 144 degrees C and incubated with primary antibodies at 60 degrees C than when heated at 95 degrees C, etched with NaIO4 and incubated with primary antibodies at 4 degrees C. Osmium-fixed IgG-gels antigen retrieved at 144 degrees C and incubated with anti-IgG at 60 degrees C showed more labelling than sections of non-osmicated gels heated at 95 degrees C. Non-osmicated gels gained significant intensity of immunolabelling when the antibody incubation occurred at 60 degrees C for 1 h than at 4 degrees C overnight. Resin embedding of pure protein gels was a useful tool for comparing different protocols for immunoelectron microscopy.

Animals↗
PubMed2002

Subcellular localization of GABA receptors in the central nervous system using post-embedding immunohistochemistry.

The following detailed protocol can be applied to demonstrate the localization of GABA receptors in CNS neurons at the ultrastructural level. While others have investigated receptors at the electron microscopic level using immunocytochemical techniques, the appearance of the tissue is usually poor and analyses of the distribution of receptors is limited. The methodology described in this paper allows for optimal preservation of the tissue while retaining immunogenicity. It does this, in part, by utilizing a balanced salt solution washout in conjunction with fixation. When the ionic composition of a fixative solution differs from extracellular fluids, like in most fixation protocols for electron microscopy, ultrastructural changes may occur in the tissue. Balanced salt solutions, like the Tyrode solution used here, helps maintain the normal extracellular environment allowing the fixing agent to reach sufficient concentration to bring about permanent and more optimal fixation even when reduced amounts of glutaraldehyde are required to preserve antigenicity. Therefore, unlike many protocols for post-embedding immunoelectronmicroscopy, this method allows for superior preservation of tissue ultrastructure compared to results previously published by others.

Animals↗
PubMed1987

The fate of embryonal-carcinoma cells in mouse blastocysts.

Double-labeled embryonal-carcinoma (ECa) cells were injected into blastocysts or incorporated into blastocysts by aggregation, and their fate after various periods of time in culture was investigated. ECa-247 cells labeled with fluorescent microscopheres were easily identified in whole blastocysts. These blastocysts were embedded in plastic, serially sectioned, and prepared for autoradiography. The 3H-thymidine label on the embryonal-carcinoma cells allowed precise localization of the cancer-derived cells. ECa-247 cells preferentially localized in the mural trophectoderm, with a few being seen in primitive endoderm and, even more rarely, in the inner cell mass. Selected autoradiograms were re-embedded and thin sectioned for transmission electron microscopy. The cancer-derived cells were found to have differentiated in accordance with their localization.

Animals↗
PubMed1996

Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells.

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.

Animals↗
PubMed1992

A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies.

A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60 degrees C to 22 degrees C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.

Biopsy↗
PubMed1992

Effects of monomeric acrylic embedding media on the antigenicity of two epitopes of the MIC2-encoded Ewing's sarcoma cell membrane antigen.

Comparative electron microscopic studies of pre- and postembedding immunolabeling experiments have shown that the antigenicity of some epitopes is lost during acrylic resin embedding of the respective tissues. In the present investigation we have tested the sensitivities of two embedding-labile epitopes (HBA-71 and HBA-45) of the Ewing's sarcoma-associated MIC2-encoded E2 antigen to the effects of the different treatment steps, which are necessary for the preparation of ultrathin sections. The extent of antigenic retention was quantitated using flow cytometry and enzyme-linked immunosorbent assays (ELISA) of tumor cell lines, thymocytes and cell membrane extracts. Fixation, dehydration and high temperature treatment of MIC2-positive cells showed only minor effects on the reactivity with the HBA-71 and HBA-45 antibodies. However, exposure of the cells to the monomeric acrylic resins LR White (LRW), LR Gold (LRG) and Lowicryl K4M at 4 degrees C for 2-18 h resulted in a significant reduction of the HBA-71 and HBA-45 reactivities. In contrast, the antigenicity of both epitopes was maintained during treatment with the apolar Lowicryl HM20 embedding medium under these non-polymerizing conditions. The resins have no direct effect on the HBA-71/HBA-45 antigen, since it could be extracted in intact form from membranes of native, but not of fixed, tumour cells using LRW for membrane solubilization. These data indicate that the HBA-71/HBA-45 antigen remains in the cell membrane and is indirectly influenced by the extraction/modification of adjacent membrane constituents. The adverse effects of the polymerization process, in the case of embedding at low temperature in Lowicryl HM20, destroyed MIC2-antigenicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic Resins↗
PubMed1993

A subgroup of bipolar cells in human retina is GABA-immunoreactive.

The distribution of GABA in the perifoveal and the near and far peripheral region of human retina was studied with peroxidase anti-peroxidase immunocytochemistry applied on semithin epoxy resin sections. Among the labeled amacrine cells in these regions, four types can be identified: putative diffuse A2 amacrines, stratified semilunar amacrines, interstitial amacrines and small displaced amacrines. GABA-immunoreactive interplexiform cells and ganglion cells also occur. Contrary to previous post-embedding studies, our preparations show that some bipolar cells in the near and far peripheral region are GABA-immunopositive. This indicates that a number of bipolar cells in human retina does have an enhanced GABA content.

Eye Enucleation↗
PubMed2006

An improved electron microscopic technique for the immunolabeling of Cryptosporidium parvum oocysts.

A technique was developed for immunolabeling Cryptosporidium parvum oocysts for subsequent observation by transmission electron microscopy. This method was developed to maintain architectural integrity of the oocyst wall and improve fixation of internal contents. The improved fixation and embedding method permits efficient immunolabeling of both nonexcysted and excysted C. parvum oocysts and may be applicable to other oocyst- and cyst-forming protozoa.

Animals↗
PubMedDate not supplied

The combination of high-accelerator epoxy resin and antigen retrieval to obtain more intense immunolabeling on epoxy sections than on LR-white sections for large proteins.

The purpose of this study was to examine how antigen retrieval affected the yield of immunogold labeling on epoxy sections based on embedding with different amounts of accelerator. The concentration of accelerator DMP-30 (tri(dimethyl amino methyl) phenol) was varied in the range of 0-8% in the processing of the tissue for epoxy embedding. Immunogold labeling was performed on epoxy sections and LR-White sections of fibrin clots and renal tissue with IgG-deposits, and the antibodies used were anti-fibrinogen anti-IgG and, respectively. For some of the sections antigen retrieval was performed by heating the sections in citrate buffer. In all cases, the yield of immunogold labeling increased following antigen retrieval. The increase (%) in the yield of immunogold labeling as a result of antigen retrieval was larger for epoxy sections than for LR-White sections. The immunolabeling on high-accelerator epoxy sections exposed to antigen retrieval was about 20% more intense than on untreated LR-White sections both for IgG and fibrinogen. In addition to breaking fixations bonds introduced by the chemical fixation, we believe that the antigen retrieval also breaks bonds between the epoxy resin and the embedded tissue. The combination of increased amount of accelerator during tissue processing for epoxy embedding and antigen retrieval by heating in citrate buffer is a potent method for increasing specific immunolabeling on epoxy sections.

Acrylic Resins↗
PubMed1994

Use of nanogold followed by silver enhancement and gold toning for preembedding immunolocalization in osmium-fixed, Epon-embedded tissues.

A reliable, sensitive, high-resolution method with good structural visualization for preembedding immunoelectron microscopy was proposed. The present technique involves the following processes. Immunolabeling of cryostat sections with primary antibodies and with nanogolds, silver intensification for visualization of nanogold secondary antibodies, gold-toning for stabilization of silver shells, and osmium postfixation and embedding in Epon for good structural visualization. The technique was applied to rat testis with anti-laminin and anti-fibronectin antibodies, showing that these two proteins are localized differentially in the lamina propria of the tissue.

Animals↗
PubMed2006

Use of anti-fluorophore antibody to achieve high-sensitivity immunolocalizations of transporters and ion channels.

We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.

Animals↗
PubMed1995

Resin embedding for quantitative immunoelectron microscopy. A comparative computerized image analysis.

Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Epon-embedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility.

Animals↗
PubMed2002

Efficient embedding technique for preparing small specimens for stereological volume estimation: zebrafish larvae.

Stereological sampling regimes, in particular volume and number estimation, often require systematic uniformly random sections throughout a specimen. A method has been developed to increase the efficiency of preparing fish larvae for sectioning prior to histological or stereological analysis. Embedding a group of larvae in a resin block using this technique greatly reduces the quantity of sections produced and allows easy assessment of sample groups. Saving time in this way therefore makes stereology a more viable research tool.

Animals↗
PubMed1995

Rapid processing of filter-grown cells for Epon embedding.

A simple and rapid method for fixation, processing, and Epon embedding of cells grown on a filter support is described. The results show that it is possible to embed filter-grown cells in resin within 30 min of applying the primary fixative and still to achieve good ultrastructural preservation.

Animals↗
PubMed1995

Postembedding alpha-tubulin immunolabelling of isolated centrosomes.

Accurate ultrastructural localization of the components of centrosomes is an important step toward the determination of their function. We have used an electron microscopy procedure to preserve centrosome-associated antigens which enables their high-resolution localization. The unique part of our procedure is the application of a post-sectioning fixation step which overcomes the poor section contrast and morphological appearance that limits the use of low-temperature processing and Lowicryl embedding. The efficacy of our approach is demonstrated by the efficient labelling of alpha-tubulin in the well-preserved and contrasted microtubule barrels of the centrides of isolated mammalian centrosomes.

Centrosome↗
PubMed2002

Immunolabeling type II collagen in the basilar membrane, a pre-embedding approach.

This paper describes the development of a protocol that can be used to detect collagen II in the healthy adult basilar membrane (BM) at the electron microscopic level. This protocol required aggressive epitope exposure techniques to break the crosslinks that bind the collagen molecules tightly into fibrils and to remove a dense mat of ground substance that surrounds the fibrils. On the other hand, the steps had to be carefully controlled to preserve BM ultrastructure and the collagen II epitopes that are typically labile. These requirements were satisfied by introducing a targeted crosslink breakage method and by regulating the duration of epitope exposure based on changes in tissue appearance observed with differential interference contrast microscopy. High levels of immunolabeling were achieved by substituting tissue preservation techniques for most or all of fixation; this was important because fixation reduces antigenicity directly and impedes epitope exposure. When these techniques were combined with more traditional trypsin and pepsin treatments, the result was dense immunolabeling and preservation of ultrastructure that allowed accurate localization of the immunolabeling. This pre-embedding immunoelectron microscopic method is the first to be carried out on the BM and may be adaptable to future studies of the BM as well as other tissues with similar molecular composition.

Animals↗