A simple technique for in situ embedding of monolayer cultures in Lowicryl K4M

A number of difficulties are encountered in embedding monolayer cultures at low temperature in Lowicryl K4M resin. In this paper we present a simple procedure using Lab-Tek Flaskettes in which fixation, processing and embedding of monolayer cultures can readily be achieved.     

Combined quick freezing,freeze-drying,and embedding tissue at low temperature and in low viscosity resins

The techniques of quick freezing and freeze-drying provide an alternative to the more classical methodologies of chemical fixation and dehydration with organic solvents. It is possible to embed freeze-dried tissue in low viscosity resins, either at room temperature or at subzero temperatures in Spurr's resin or Lowicryl K4M, respectively. The choice of embedding medium affords additional flexibility in postdrying and embedding conditions, since Spurr's resin allows vapor fixation with osmium tetroxide and thermal polymerization. Osmium tetroxide is not recommended for Lowicryl resins, but these media permit polymerization at subzero temperatures with ultraviolet light. Both resins have unique advantages that may be utilized, depending upon the purpose of the embedding. In this paper, we discuss the details of preparing smooth muscle, from rabbit renal artery, by quick freezing and freeze-drying, as well as methods for the embedding of the freeze-dried tissue in both Spurr's resin and Lowicryl K4M. Although we have previously reported the ultrastructure of smooth muscle embedded in Spurr's low viscosity resin, the combination of freeze-drying and infiltration in Lowicryl K4M represents a new approach that allows the elimination of chemical fixation, dehydration with organic solvents, and heat polymerization of the embedding medium.     

Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro

The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as (1) undifferentiated multilayers in glucose-containing culture media and (2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins. Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; “pre-differentiated” columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface.     

Extraction of membrane lipids during fixation,dehydration and embedding of Acholeplasma laidlawii-cells for electron microscopy

The aim of the present investigation was to study the extent to which lipids are extracted from biological membranes during dehydration and embedding procedures carried out at high or low temperatures. Cells of Acholeplasma laidlawii were used as experimental material, since the lipids of this bacterium easily can be radioactively labelled without labelling the rest of the cell, and the lipids are almost entirely located in the cytoplasmic membrane. The cells were fixed at 277 K with glutaraldehyde, sequentially with this reagent and osmium tetroxide, or with glutaraldehyde, osmium tetroxide and uranyl acetate in that order. Loss of lipid during these procedures was negligible. When cells fixed with glutaraldehyde and osmium tetroxide were dehydrated with ethanol at room temperature and embedded in Epon at 333 K, i.e. subjected to a conventional treatment, about 90% of the lipid content of the cells was extracted. The loss was reduced to c. 20% when treatment with uranyl acetate was included in the procedure and the non-polar methacrylate resin Lowicryl HM20 was substituted for Epon. When cells fixed with glutaraldehyde and osmium tetroxide were dehydrated with ethanol at 238 K and embedded in Lowicryl HM20 at room temperature, practically no lipid was extracted. Substitution of the polar methacrylate-acrylate resin Lowicryl K4M for Lowicryl HM20 resulted in the loss of about half of the lipid content of the cells. The use of ethanediol as dehydrating agent instead of ethanol did not diminish the extraction. Cells fixed solely with glutaraldehyde lost about half of their lipid content, even when both dehydration and embedding was performed at 238 K. The lipid material extracted from glutaraldehyde-fixed cells contained slightly more saturated fatty acids than that remaining in the cells. The reverse was true for osmium tetroxide-fixed cells. With respect to lipid species, the extractions were generally rather unspecific.     

Freeze-substitution and low-temperature embedding of dairy products for transmission electron microscopy

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze‐substituted and embedded at low temperature (?20 °C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non‐dairy mixed systems were frozen by plunging in propane, at its boiling point (?187 °C). Ice cream, because it is already frozen, was fractured into 1‐mm³ pieces in liquid nitrogen and then added to frozen fixative (?196 °C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step‐wise from ?80 to ?20 °C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non‐polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at ?20 °C using ultraviolet light to cross‐link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.     

Low temperature embedding with Lowicryl resins: two new formulations and some applications

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.     

Immunofluorescence and protein A-gold techniques in localization of plant pathogen antigens in Lowicryl K4M embedded tissue

This paper investigates the use of Lowicryl K4M in the embedding of apple tissue for immunocytochemistry. The localization of the extracellular protease of the apple pathogen, Nectria galligena Bres., in infected apple tissue by immunofluorescence and protein A-gold immunoelectron microscopy is also described. Infected apple tissue was fixed in 0.5% glutaraldehyde and 4% paraformaldehyde and embedded in Lowicryl K4M at 313 K. The protease was isolated and purified from rotted apple tissue by gel filtration and ion-exchange chromatography. Monospecific antibodies against the protease were raised in rabbits and purified by protein A-affinity chromatography. Incubation of apple tissue sections, infected with N. galligena, with the mono-specific antibody and tetramethylrhodamine isothiocyanate (TRITC)-conjugate, resulted in specific fluorescence of fungal hyphae and cytoplasm of apple cells. Similar localization of colloidal gold particles over hyphae and host cell cytoplasm was demonstrated employing protein A-gold immunochemistry. The low temperature embedding resin Lowicryl K4M appears to provide adequate morphological preservation of apple tissue and excellent retention of antigenicity. TRITC conjugates and protein A-gold may prove useful in immunocytochemical investigations of plant-pathogen interactions.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Developments of new Lowicryl® resins for embedding biological specimens at even lower temperatures

Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210K (hydrophilic K11M) and to 190K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the ‘progressive lowering of temperature’ procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo-polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.     

Optimal preparatory procedures of cryofixation for immunocytochemistry

To examine the optimal preparatory procedures of cryofixation for immunocytochemistry, the labeling density over the antigenic sites in cells processed by various protocols of freeze-substitution and embedding was quantitatively evaluated. Fresh tissue blocks of gerbil parotid gland were quickly frozen by a metal contact method using liquid helium and freezesubstituted with one of the following media: 4% OsO₄ in acetone or 0.4% OsO₄ in acetone or 0.3% glutaraldehyde in acetone. They were then embedded in either an Epon-Araldite mixture or Araldite 6005, which were polymerized at 60°C and 50°C, respectively. Some frozen samples substituted with aldehyde-containing acetone were embedded in Lowicryl K4M (polymerized at —30°C). Immunocytochemical localization of amylase was examined by indirect immunostaining by using antigerbil parotid amylase antibody and protein A/gold complex. Thin sections of epoxyresin-embedded materials were treated with oxidizing agents before immunostaining. The central dense core of heterogeneous secretory granules in the acinar cells was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The labeling density on thin sections of all the cryofixed materials examined was about 1.5 times or more as high as in those processed by conventional chemical fixation. The highest value of the labeling density was obtained from material which was substituted with 0.3% glutaraldehyde in acetone and embedded in Araldite 6005. Substitution with osmium-containing acetone appeared not to seriously affect immunoreactivity of the antigenic sites and was advantageous because of the distinctive images of membranes. Advantages and disadvantages of the individual protocols employed are discussed.     

High-pressure freezing for immunocytochemistry

Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross-linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample-stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well-frozen material obtainable to ≈ 15 μm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystal-artefact-free freezing of samples up to 200 μm in thickness. There have been few reports of high-pressure freezing for immunocytochemical studies and there is no consensus on the choice of post-freezing sample preparation. A range of freeze-substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin-embedding, polymerization and immunocytochemical labelling. Freeze-substitution in acetone containing 2% osmium tetroxide followed by epoxy-resin embedding at room temperature gave optimum morphology. Freeze-substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl-embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze-substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice-crystal damage in sections of embedded material. Thus a slow warm-up to freeze-substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze-substitution media from immunocytochemical labelling experiments.     

The control of temperature during polymerization of Lowicryl K4M: there is a low-temperature embedding method

Methods are described for controlling the temperature of Lowicryl K4M in flat embeddings and in capsules during polymerization under ultra-violet (UV) irradiation in an Agar UVF 35 low-temperature cabinet. Aluminium blocks or an ethanol bath are used as heat ‘sinks’. Consequently the question posed by Ashford et al. (1986) — “Is there a low-temperature embedding method?” — is answered in the affirmative. This control of temperature is particularly important when using gelatin capsules since a temperature rise of as little as 2 K results in uneven polymerization.     

Silver enhancement of Nanogold particles during freeze substitution for electron microscopy

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well‐fixed, well‐resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno‐localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron‐dense markers, like Nanogold®, will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor‐mediated membrane traffic.     

Lipid losses during processing of cardiac muscle for electron microscopy

The lipid retaining properties of several methods of processing tissue for electron microscopy (EM) have been assessed quantitatively. Guinea-pig hearts were perfused in vitro at 37°C with ³H-oleic acid bound to albumin. The hearts were fixed by perfusion with 4% glutaraldehyde in 0.1 M cacodylate buffer. Pieces of left ventricle and interventricular septum were removed., weighed and processed for EM. The fluids used at each stage of processing were monitored for loss of radioactive lipid by scintillation counting. Lipids were extracted from the processed tissue immediately before the embedding stage using a mixture of chloroform: methanol (2:1 v/v). Counts from processed tissue were compared with counts from tissue extracted directly after perfusion fixation in order to monitor subsequent losses during processing. A modified version of Epon processing, omitting 100% ethanol, acetone or propylene oxide, gave a lipid retention of only 20.6%. The addition of para-phenylenediamine to the procedure did not improve the retention although this has been shown to be a useful stain for intracellular lipid. Water soluble Durcupan which does not involve ethanol or acetone dehydration has an average retention of 63% with 100% recovery while the lesser known polymer GACH, a mixture of glutaraldehyde and carbohydrazide used both for dehydration and embedding showed a lipid retention of 82% of the counts recovered although recovery was only 69%. An attempt was made to determine which classes of lipids were present in the tissue after perfusion fixation using thin layer chromatography. It was found that the presence of any of the processing fluids affected the polarity of the lipids and their rates of migration on thin layer plates.     

Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens

The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.     

Dimensional changes of cultured smooth muscle cells due to preparatory processes for transmission electron microscopy

The change in volume of cultured smooth muscle cells prepared with four different fixation procedures for transmission electron microscopy was studied. Although the cells showed swelling after being embedded in Spurr's embedding medium, the degree of swelling depended on the particular method of fixation procedure used. When the volume of the cells measured using transmission electron microscopy was compared with that of the fresh cell volume, cells prepared by two of the methods showed swelling, and cellular shrinkage was noted in the other two methods. One method which caused the least amount of volume change is recommended for quantitative electron microscopic study of vertebrate smooth muscle cell systems.     

Why low temperature embedding for X-ray microanalytical investigations?

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to ?50°C. Sections of low temperature embedded material can be cut dry at ?60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze-dried and embedded tissue.     

Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

We describe a method for high‐pressure freezing and rapid freeze‐substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed ‘face to face’ without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice‐crystal artefact. Freeze‐substitution at −90°C for 60 min in acetone containing 2% uranyl acetate, followed by warming to −50°C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.     

A double lead stain method for enhancing contrast of ultrathin sections in electron microscopy: a modified multiple staining technique

A modification of the conventional method for the staining of ultrathin sections resulted in an increase in contrast of ultrastructural detail in tissues. Tissues embedded in Spurr's low viscosity embedding medium were stained with freshly centrifuged Reynolds' lead citrate for 1–5 min, rinsed in double distilled water and dried prior to staining with a saturated solution of uranyl acetate for 40 min, and freshly centrifuged Reynolds' lead citrate for 20 min. Sections treated by this procedure showed enhanced staining of cellular organelles and cytoplasmic matrix. This procedure is recommended for tissues with poor staining qualities resulting from either prolonged fixation or from inadequacies in the buffer or embedding medium used.     

Temperature control in Lowicryl K4M and glycol methacrylate during polymerization: is there a low-temperature embedding method?

An apparatus for embedding tissues at resin temperatures down to 228 K is described. By placing thermocouples in the resin the temperature has been monitored during embedding at low temperature with glycol methacrylate (GMA) and Lowicryl K4M. Even in this apparatus with a liquid cooling bath the heat of polymerization is not dissipated and the resin temperature rises. This rise is directly proportional to the resin temperature at the onset of polymerization and is higher in Lowicryl K4M than GMA. The initial resin temperature also affects the time taken for polymerization. The time to the onset of the peak and its duration are both increased as the temperature is lowered. This effect is more pronounced with GMA than Lowicryl K4M and polymerization of GMA is inhibited at the lowest temperature used. When Lowicryl K4M, polymerized at low temperature, is warmed up to ambient a further exothermic reaction occurs, which causes the resin temperature to rise well above ambient. Both this temperature peak and that during polymerization are reduced, but not totally eliminated, by reducing the resin volume. Aircooled systems are inefficient compared with the low-temperature apparatus used here and the resin temperature rise is consequently greater and, even with small resin volumes, it can be very high. It is therefore unlikely for published methods that the temperature specified has been maintained in the resin during polymerization. The implications of these findings are discussed in relation to enzyme and antigen survival. Recommendations include use of very small volumes of resin, refrigerated liquid-bath rather than air-cooled systems and contact with a heat sink when specimens are warmed up to ambient temperature. Examples of enzyme reaction, antigen survival and structural preservation obtained with the method are presented.