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     

Enhanced ultrastructural preservation of cartilage proteoglycans in the extended state

We investigated the preservation of proteoglycan (PG) structure in rat epiphyseal cartilage using N-N-dimethylformamide (DMF) dehydration before embedding. After aldehyde fixation, specimens with and without routine osmium post-fixation were dehydrated in graded DMF and embedded in either Spurr's resin or Lowicryl K4M resin. Standard ethanol dehydration with Spurr or Lowicryl embedding techniques resulted in the formation of condensed PGs, called matrix granules. DMF dehydration before embedding greatly improved the preservation of PG structure and resulted in an extended appearance of PGs closely resembling the fine filamentous network of cartilage tissues processed by rapid freezing and freeze-substitution. However, en bloc staining of aldehyde-fixed specimens with cationic reagents before or during DMF dehydration induced the condensation of PGs and resulted in the formation of matrix granules. These observations demonstrate that DMF, a mild dehydration agent, dramatically improves PG preservation without a harmful effect on aldehyde-fixed PG structure and can be utilized regardless of routine post-fixation.     

The use of osmicated tissues for Lowicryl K4M embedding.

Tissue chopper slices of rat incisor, rat parotid gland, and chicken tibiae, fixed with 1% glutaraldehyde, were post-fixed with potassium ferrocyanide-reduced osmium tetroxide, dehydrated with methanol, and conventionally embedded in Lowicryl K4M at -20 degrees C. The tissues showed an ultrastructural appearance comparable with that of their Epon-embedded counterparts and, in particular, the Golgi apparatus was well defined. Furthermore, Lowicryl K4M-embedded osmicated tissues permitted both post-embedding lectin-gold cytochemistry and immunogold labeling.     

Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.     

Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures.

The intracellular location of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris was determined using pre-embedding and post-embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One-day-old cultures of D. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post-embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low-temperature resin Lowicryl K4M. Our post-embedding studies using antibody-gold or protein-A-gold as electron-dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre-embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin-linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.     

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.     

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     

Contrasting of Lowicryl K4M thin sections

Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Contrasting of Lowicryl K4M thin sections.

A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.     

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     

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.     

Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.     

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.     

Glycoconjugate cytochemistry of the rat fundic gland using lectin/colloidal-gold conjugates and Lowicryl K4M. Helix pomatia lectin is a specific marker for mucous neck cells in fundic glands of the rat gastric mucosa

The fundic gland of the rat stomach was studied using the low-temperature embedding resin Lowicryl K4M and postembedding staining with lectin/colloidal-gold (CG) conjugates. Intense labeling with Ricinus communis agglutinin I was observed not only in mucous-producing cells but also in parietal cells. In contrast, Helix pomatia agglutinin (HPA) only labeled mucous neck cells and intermediate cells between mucous neck cells and chief cells. The other epithelial cells present in the rat fundic gland showed virtually no reaction with this lectin. Our results indicate that HPA might be a marker lectin of mucous neck cells and their derivatives. The combination of embedding in the hydrophilic resin Lowicryl K4M and postembedding staining with lectin-CG conjugates provided satisfactory staining results, and made it possible to visualize the precise distribution of terminal glycoconjugates in intracellular components as well as on the plasma membrane.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes

Involucrin immunoreactivity was localized ultrastructurally with protein A--gold in epidermis and cultured keratinocytes embedded in Lowicryl K4M. In the skin, immunoreactivity was found predominantly in cells of the granular layer and inner stratum corneum. The label was associated primarily with amorphous cytoplasmic material and especially keratohyaline granules. Some labeling was observed at the cell periphery, but little with keratin filaments. Tissue samples examined without aldehyde fixation showed relatively greater labeling in the outer stratum corneum than fixed tissue. In cultured cells, the labeling was also associated primarily with cytoplasmic granular material and to a lesser extent with the cell periphery. Upon treatment with the ionophore X537A, keratin filaments were found in aggregated arrays and the plasma membranes became convoluted. That involucrin immunoreactivity persisted in the cytoplasm in cultured cells and in vivo after cross-linking occurs could account for considerable isopeptide bonding detected in epidermal keratin fractions and indicates that not all the involucrin participates in envelope formation.     

Glycoconjugate cytochemistry of the rat fundic gland using lectin/colloidal-gold conjugates and Lowicryl K4M

Summary The fundic gland of the rat stomach was studied using the low-temperature embedding resin Lowicryl K4M and postembedding staining with lectin/colloidal-gold (CG) conjugates. Intense labeling with Ricinus communis agglutinin I was observed not only in mucous-producing cells but also in parietal cells. In contrast, Helix pomatia agglutinin (HPA) only labeled mucous neck cells and intermediate cells between mucous neck cells and chief cells. The other epithelial cells present in the rat fundic gland showed virtually no reaction with this lectin. Our results indicate that HPA might be a marker lectin of mucous neck cells and their derivatives. The combination of embedding in the hydrophilic resin Lowicryl K4M and postembedding staining with lectin-CG conjugates provided satisfactory staining results, and made it possible to visualize the precise distribution of terminal glycoconjugates in intracellular components as well as on the plasma membrane.     

Immunocytochemistry on fine needle aspirates in paraffin miniblocks

A simplified method of processing of fine needle aspirates for paraffin miniblocks suitable for both morphologic and immunocytochemical evaluation is described. Aspirates were fixed in ethanol at 4 degrees C, dehydrated in acetone and xylene and embedded in paraffin (58 degrees C). All steps were carried out in a single Eppendorf centrifuge tube; the total process took less than four hours. Deparaffinized sections were stained using the alkaline phosphatase-antialkaline phosphatase technique with monoclonal and conventional antibodies helpful in the differential cytologic diagnosis of alcohol-fixed aspiration biopsy specimens. Antibodies to keratin, vimentin, desmin, neurofilaments, glial fibrillary acidic protein, leukocyte-common antigen, synaptophysin and immunoglobulin kappa and lambda light chains reacted positively on the miniblock material. Since the paraffin miniblocks combine the histologic pattern of the tumor with the differentiation-specific information provided by immunocytochemistry, their use can improve the accuracy of tumor typing in aspirates.     

The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.