A Multichromatic Stain for Lowicryl K4M Embedded Tissues

A staining procedure using celes-tine blue and chrome-trope 2R for Lowicryl K4M embedded tissues is presented. The stain produces a reliable multichromatic stain for light microscopy on Lowicryl embedded semithin 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.     

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.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

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.     

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.     

Immunocytochemical demonstration of serine:pyruvate aminotransferase in peroxisomes and mitochondria of rat kidney

Summary The light- and electron-microscopic localization of serine:pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Immunocytochemical demonstration of serine: pyruvate amino-transferase in peroxisomes and mitochondria of rat kidney

The light- and electron-microscopic localization of serine: pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.     

Immunoelectron microscopical localization of ornithine transcarbamylase in hepatic parenchymal cells of the rat

Summary The electron microscopical localization of ornithine transcarbamylase in rat liver was investigated by a protein A—gold technique applied to thin sections of Lowicryl K4M- or LR gold-embedded materials and to ultracryosections. Gold particles were exclusively confined to mitochondria of the parenchymal cells but not of sinus-lining cells. In mitochondria, gold particles were present in the matrix and closely associated with the inner membrane. The most intensive labelling was obtained from ultracryosections, while weaker labelling was noted in sections of materials embedded in both Lowicryl K4M and LR gold. The association of the enzyme with the inner membrane was confirmed by quantitative analysis of distribution pattern.     

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.     

Lowicryl K4M embedding of brain tissue for immunogold electron microscopy

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.     

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.     

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.     

Post embedding immunoelectron microscopy of human breast cancer: a comparison of three acrylic resins

Summary The suitability of three acrylic resins for the immunoelectron microscopical localization of cell surface and cytoskeletal antigens in surgically excised, immersion fixed human breast cancer, using an immunogold system, has been assessed.Good localization of milk fat globule membrane was achieved with LR White, LR Gold and Lowicryl K11M, although the embedding schedule for LR White had to be modified. The best results were achieved with Lowicryl K11M. Only scanty labelling of actin and cytokeratin was seen in LR White embedded tissue, whereas there was clear localization in LR Gold and Lowicryl K11M embedded samples. Tubulin and -actinin was detected at low level in tissues in the low temperature embedding resins, but not in LR White embedded samples. The morphology of the latter was poorer, and there was greater variability in ultrastructure and labelling.Of the two low temperature embedding resins, Lowicryl K11M gave slightly better results. However, the advantages could be outweighed by the problem incurred in achieving the low temperatures, and by poorer handling properties than LR Gold.     

Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M

Summary In this study we present a postembedding on-grid immunogold labelling procedure for the ultrastructural localization of the HIV-1 core protein p24. HIV-1 infected cells were fixed in 0.1% glutaraldehyde, incompletely dehydrated and embedded in LR White or in Lowicryl K4M. Antigenic sites were detected by incubation of ultrathin sections with primary mouse monoclonal antibody anti-HIV-1 p24, followed by the secondary antibody goat anti-mouse IgG coupled to 10nm gold particles. Antigenicity of p24 was found to withstand the applied fixation and was shown to be preserved in LR White as well as in Lowicryl. The described procedure permits the uncomplicated and easy detection of p24 in HIV-1 infected cells and tissues.     

Immunocytochemical localization of enterobacterial common antigen in Escherichia coli and Yersinia enterocolitica cells.

Enterobacterial common antigen (ECA) was localized on Lowicryl K4M sections and on ultrathin cryosections by using either a mouse monoclonal antibody or an absorbed rabbit polyclonal immune serum with the corresponding gold-labeled secondary antibodies. Comparable results were obtained with both monoclonal antibody and polyclonal immune serum. Controls with two ECA-negative mutants revealed the ECA specificity of both labeling systems. On Lowicryl K4M sections, good labeling of the outer membrane and of membrane-associated areas in the cytoplasm was obtained. Unexpectedly, however, the ribosome-containing areas of the cytoplasm also showed significant labeling. On ultrathin cryosections, labeling of the cytoplasmic areas was much weaker, although the density of label in the outer membrane was comparable to that obtained with the Lowicryl K4M sections. With the techniques used, it cannot be completely excluded that the appearance of ECA in the cytoplasm is due to displacement of ECA-reactive sites during the preparation procedure.     

Post-embedding methods for immunolocalization of elastin and related components in tissues

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.     

Applications of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections

Previous studies have demonstrated that antigens or lectin-binding sites can be localized in sections from paraffin-embedded tissues with protein A or lectins bound to colloidal gold or colloidal silver (Roth J: J Histochem Cytochem 30:691, 1982 and 31:547, 1983). In the present study the protein A-gold technique and lectin-gold complexes have been applied to semithin sections (0.5-1.5 micron) of Epon- or low temperature Lowicryl K4M-embedded rat pancreas, kidney and submandibular gland. The results show that an increase in resolution and, therefore, in amount of information can be obtained. The optimal mode of imaging was determined on sections without counterstaining. Bright-field illumination gives the maximum information about the staining signal, while phase-contrast and Nomarski differential interference contrast give predominantly structural and, to a lesser extent, staining information. Polarization epi- and transillumination microscopy is inferior in all aspects. The application of a battery of lectin-gold complexes to rat submandibular gland revealed a specific staining pattern for each lectin in acinar and excretory duct cells.     

A new acrylic resin formulation: a useful tool for histological, ultrastructural, and immunocytochemical investigations.

We describe a new formulation for a hydrophilic resin, mostly composed of glycol methacrylate and hydroxypropyl methacrylate and here referred to as bioacryl, that allows the performance of morphological and immunohistochemical investigations at both light and electron microscopic levels. Immunolocalizations performed on bioacryl-embedded tissues are characterized by high specificity with virtually absent background staining. Finally, the new resin yields satisfactory fine-structural preservation, resulting in ultrastructural images of better quality than those obtained with Lowicryl K4M.