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.     

Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.     

A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.     

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.     

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO₄ fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.     

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO₄ fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.     

Use of LR white resin for post-embedding immunolabelling of brain tissue.

The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.     

Development of a Plastic Embedding Method for Large-Volume and Fluorescent-Protein-Expressing Tissues

Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.     

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.     

Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding

Summary In the present study the cellular fine structure of freeze-substituted sporidia of the phytopathogenic fungusUstilago avenae is investigated by means of thin-section electron microscopy. A conventional embedding method using Spurr's low viscosity resin is compared with the recently developed methacrylate mixtures Lowicryl^® K 4 M and HM 20 resin. Generally, freeze-substitution yields improved preservation of fine structural details of the fungus compared to previously applied conventional fixation methods. Using double fixation during freeze-substitution prior to conventional embedding the fungal membrane system (plasmalemma, endoplasmic reticulum), organelles (mitochondria, nucleus etc.) and other cytoplasmic features (ribosomes, cytoskeleton) appear well resolved and smoothly contoured. Aldehyde fixed and Lowicryl embedded sporidia ofU. avenae resemble these double fixed fungal specimens fairly closely. The complete low-temperature preparation produces visualization of distinct cellular details although contrast reversal of cellular membranes (er, mitochondria etc.) is sometimes observed. In particular, fine structure resolution is enhanced in Lowicryl HM 20 embedded fungal cells. This is due also to significant improvement in staining of the cellular membranes, cytoskeleton (microfilaments and microtubules) and Golgi apparatus-like areas, using tannic acid. In case of the fungusU. avenae, freeze-substitution in combination with mild glutaraldehyde fixation and final low-temperature embedding in HM 20 resin prove suitable for improved preservation of cellular ultrastructure.Abbreviations cw cell wall - cy cytoplasm - FS freeze-substitution - FS-A GA/OsO₄ freeze-substitution and Spurr's LV-embedding - FS-B GA freeze-substitution and Lowicryl K 4 M LT-embedding - FS-C GA freeze-substitution and Lowicryl HM 20 LT-embedding - go Golgi apparatus-like body - GA glutaraldehyde - g glycogen deposit - l lipid droplet - LT low temperature - Lowicryl LT-embedding Lowicryl low-temperature embedding - Lowicryl LT-resin Lowicryl low-temperature resin - MeOH methanol - mf microfilament - mt microtubule - m mitochondrion - mvb multivesicular body - ne nuclear envelope - np nuclear pore - npl nucleoplasm - nu nucleolus - n nucleus - OsO₄ osmium tetroxide - pl plasmalemma - pr polyribosomes - Pb-citrate Reynolds' lead citrate - r ribosome - RT room temperature - rer rough endoplasmic reticulum - Spurr's LV-embedding Spurr's low viscosity embedding - Spurr's LV-resin Spurr's low viscosity resin - t tonoplast - Uac uranyl acetate - v vacuole     

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.     

Comparison of LR white resin,lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

Summary The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40°–50° C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.     

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.     

An improved ultrastructural double-staining method for rat growth hormone and its mRNA using LR White resin: a technical note

An improved new method for the simultaneous visualization of mRNA and encoded protein in LR White resin-embedded specimens is described. This pre-embedding electron microscopical in situ hybridization (procedure) localized rat growth hormone mRNA specifically as high electron-density products on the polysomes of the rough endoplasmic reticulum. A subsequent post-embedding immunoreaction, using protein A colloidal gold particles, identified growth hormone as gold particles both in the cisternae of the rough endoplasmic reticulum and on the secretory granules. In our previous report, we used Epon resin for tissue embedment, which required an etching process using hydrogen peroxide or sodium periodate for immunoreactivity retrieval. In general, osmification and embedment in Epon resin are reported to decrease the immunoreactivity of the targeted protein, and the etching process using hydrogen peroxide or sodium periodate results in deosmification and shades off the signals of mRNA. To resolve these problems, we have recently used LR White resin for tissue embedment. In LR White resin-embedded tissues, retrieval of immunoreactivity using hydrogen peroxide or sodium periodate is not required, and, therefore, the gradation of the signals of mRNA can be avoided. © 1998 Chapman & Hall     

Comparison of LR white resin, Lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.     

Immunoelectron microscopic demonstration of thyroglobulin and thyroid hormones in rat thyroid gland

We have developed a post-embedding immunogold technique for electron microscopic localization and quantitation of thyroglobulin (TG), thyroxine (T4), and triiodothyronine (T3) in rat thyroid. Labeling for TG was located on rough endoplasmic reticulum, Golgi apparatus, exocytotic vesicles, luminal colloid, colloid droplets, and lysosomes, whereas labeling for thyroid hormones was located on luminal colloid, colloid droplets, and lysosomes. We tested different procedures of fixation, dehydration, embedding, polymerization, and immunoincubation to optimize ultrastructural preservation and immunolabeling. Fixation with glutaraldehyde and osmium was possible with retained antigenicity. Dehydration temperature and the choice of embedding resin were the two crucial factors for good immunolabeling. Low-temperature dehydration greatly improved immunolabeling and could be combined with embedding in the methacrylate LR White or the epoxide Agar 100 (equivalent of Epon 812) polymerized at 40-60 degrees C, as the temperature during subsequent embedding and polymerization was of little importance for the immunoreactivity. Labeling on LR White sections was always higher than on Agar 100 sections. Various etching procedures were tested without improved specific labeling. Etching with hydrochloric acid gave nonspecific labeling of certain cell compartments.     

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.     

Parallel monitoring of RNA abundance,localization and compactness with correlative single molecule FISH on LR White embedded samples

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.     

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.     

Influence of embedding media in prolactin labelling with immunogold techniques

Summary Immunogold labelling of prolactin in three different embedding media was compared. The polymeric prolactin in secretory granules was labelled in the three media, however, acrylic monomers (Lowicryl K4M and LR White) provided a more intense labelling with higher dilutions of the primary antibody, compared to the labelling in the epoxy resin (araldite). An intense labelling of monomeric prolactin in Golgi complex was detected only in acrylic embedments, and the labelling on the rough endoplasmic reticulum was significant only in LR White embedded tissues.