LR White is preferable to Unicryl for immunogold detection of fixation-sensitive nuclear antigens

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.     

The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method

A methodology is described in which a number of well-established research techniques are brought together to enable the complete diagnostic analysis of a renal biopsy on a single piece of tissue. By embedding the biopsy in the acrylic resin LR White, unsupported sections of which are stable in the electron beam, light and electron microscopy and immunocytochemistry become feasible on sections from the same block. The biopsy is glutaraldehyde fixed but post-fixation in osmium tetroxide, which is often deleterious to antigen preservation, is omitted. Extraction in organic solvents and resin monomer is minimized by rapidly infiltrating the tissue from 70% ethanol and polymerizing the resin catalytically at 0 degrees C. Semithin sections can be stained with haematoxylin and eosin, Toluidine Blue or methenamine silver, giving results similar or superior to those obtained from paraffin sections. Thin sections show that the standard of morphological preservation is similar to that seen using epoxide sections even though the kidney is unosmicated. The tissue retains a high level of antigen reactivity, which, in the limited number of cases so far examined, has paralleled or exceeded that demonstrated by conventional immunofluorescence on frozen sections.     

Characterization of glycoconjugate antigens in mouse embryonic neural precursor cells

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified beta1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.     

Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections.

We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.     

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.     

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold

The introduction of acrylate resins (Lowicryl K4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.     

Two Methods for Flat Embedding Sections in LR White Cut from Paraffin Blocks

Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistr y.     

Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0°C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.     

Ultrastructural immunocytochemistry of glia cells

Summary The introduction of acrylate resins (Lowicryl K 4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class. H antigens by pre-embedding method then followed by post-embedding IGS for the other intracytopasmic antigens.The Clinical Research Unit for Multiple Sclerosis is supported by Hermann and Lilly Schilling foundation     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

LR White embedding allows a multi-method approach to the analysis of brain tissue from patients with Alzheimer''s disease

Summary Light (LM) and electron (EM) microscope comparisons of the cytochemistry and immunocytochemistry of neuritic plaques and neurofibrillary tangles, the main histopathological changes in the brains of Alzheimer's disease sufferers, have been almost impossible because of the disparity between the two technologies. By embedding unosmicated brain tissue in the acrylic resin LR White, direct comparisons can be made between techniques applied at the LM level with those at the EM level.After partial dehydration in 70% ethanol, the tissue is embedded by rapid infiltration and polymerization at 0°C, which has been shown to maximally preserve tissue immunoreactivity. Semithin sections are then receptive to routine LM stains, silver stains e.g. Gomori's methenamine silver, and immunocytochemistry with immunoperoxidase or immunocolloidal gold. Serial thin from the use of a mouse monoclonal antibody against -amyloid and a rabbit polyclonal antibody against ubiquitin are presented. LR White resin includes no elements other than carbon, oxygen and nitrogen, of which it is composed, so that sections of it are valuable for sensitive X-ray energy dispersive microanalysis.     

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.     

Improvement of prolactin immunolabelling in osmium-fixed acrylic-embedded pituitary gland

Immunolabelling of prolactin (PRL) with protein A-colloidal gold complex and tissue fine structure were enhanced after postfixation of pituitary gland with osmium tetroxide and embedment in acrylic momers (LR White). Thin sections were treated with sodium metaperiodate before immunocytochemistry. An intense PRL labelling was detected in secretory granules, Golgi complexes and extracellular accumulation of the hormone. The use of osmium greatly improved the fine structure of the tissue and its stability during acrylic embedment.     

Enhancement of the periodic acid--Schiff (PAS) and periodic acid--thiocarbohydrazide--silver proteinate (PA-TCH-SP) reaction in LR white sections

LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination: 1) The PAS reaction in semithin sections turns out stronger after partial (70% ethanol) than complete (100% ethanol) dehydration of the tissue before its transfer to 100% LR White. 2) Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end. 3) The use of hot silver proteinate (50 degrees C) plus strong silver enhancement (15-20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.--Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.     

Immunoregulatory activity of human bone marrow. Identification of suppressor cells possessing OKM1, SSEA-1, and HNK-1 antigens

Human bone marrow contains natural regulatory cells capable of suppressing the in vitro primary IgM response of normal tonsillar cells. The suppression is mediated by non-T cells possessing Fc receptors, OKM1, SSEA-1, and HNK-1 antigens on their surface. The suppression was abrogated by treatment of bone marrow cells (BMC) with anti-HNK-1 or anti-SSEA-1 antisera and complement. Furthermore, BMC depleted of HNK-1+ cells could respond in a primary in vitro antibody response when provided with accessory T cells and macrophages from tonsillar cells. Our findings support the idea that HNK-1+ and HNK-1- BMC populations act antagonistically in the regulation of antibody synthesis. Further, the finding of HNK-1+, SSEA-1+, and OKM1+ suppressor cells in human bone marrow may represent a precursor phenotype of mature natural killer cells with potent immunoregulatory activity.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies

Summary A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies.

A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

Immunocytochemistry of contractile and cytoskeletal proteins in smooth muscle: Lowicryl,LR White,and polyvinylalcohol compared

Summary Three embedding media have been compared with respect to post-embedding immunolabeling of contractile and cytoskeletal antigens in aldehyde-fixed smooth muscle tissue: the methacrylate derivates lowicryl K4M (cured at –35 or 60°C) and LR White (cured at 0 or 60°C) and the water soluble resin, polyvinylalcohol (dried at 60°C). Measurements of intensity of labeling of ultrathin sections in the fluorescence microscope showed that five antigens (actin, myosin light chain, tropomyosin, filamin and vinculin) reacted more or less equally with their respective antibodies in all the embedding media, including those cured at 60°C. One antibody (anti-light meromyosin) reacted well only with polyvinylalcohol-embedded tissue. In contrast to the relative invariance of antibody reactivity between media clear differences in the preservation of ultrastructural integrity were observed. Embedding in polyvinylalcohol (dried at 60°C) and in Lowicryl (cured at –35°C) resulted in superior preservation as compared to Lowicryl or LR White cured at 60°C. Examples of uitrastructural immunocytochemistry with the antibodies against filamin and myosin light chain, using the immunogold staining procedure are presented: the sites of localization by these antibodies were the same with all the media tried. The relative merits of the different methods are discussed.Abbreviations EGTA Ethyleneglycol-bis(-amino ethyl ether)N,N,N,N-tetra acetic acid - PIPES 1,4-Piperazinediethanesulfonic acid - LR London Resin     

Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.