Modification of the Falck-Hillarp formaldehyde fluorescence method using the vibratome®: simple,rapid and sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue

Summary Two modifications of the original Falck-Hillarp formaldehyde fluorescence technique are presented, both based on a recently introduced instrument, the Vibratome^®, which permits cutting of unembedded tissue with a section thickness down to 10 ,.The first modification involves sectioning of unfixed tissue at a temperature below +5°C, subsequent air drying and reaction with formaldehyde vapours. In the second procedure formalin fixed tissue is cut and processed as described above. It is essential that both formalin fixation and cutting of the fixed tissue takes place at a low temperature to avoid diffusion of the catecholamines.The results show that with both techniques central CA neurons can be visualized with a high degree of sensitivity. Furthermore, since the sections are free from fractures—a common problem in freeze-dried tissues—the Vibratome^® technique represents a valuable tool for mapping studies. It may also be added that since many steps of the original procedure are omitted the present techniques are also more rapid and simple. It is pointed out that using the Vibratome^® procedure on formalin fixed tissue, it will be possible to combine e.g. cholinesterase staining or Fink-Heimer silver impregnation or immunofluorescent studies with the Falck-Hillarp technique on serial or even on the same sections.     

Application of the aluminum-formaldehyde (ALFA) histofluorescence method for demonstration of peripheral stores of catecholamines and indolamines in freeze-dried paraffin-embedded tissue,cryostat sections and whole-mounts

Summary This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.     

Verwendung gefriergetrockneter Kryostatschnitte für histologisehe und histochemische Untersuchungen

Zusammenfassung Die Anwendungsmöglichkeiten verschiedener histologischer und histochemischer Techniken an gefriergetrockneten Kryostatschnitten wird beschrieben. Es wird gezeigt, wie die Schnitte auf Objektträgern montiert und in wässrige Medien eingebracht werden können. Dabei treten nach kontrollierter Gefriertrocknung weit weniger Artefakte auf als bei Weiterverarbeitung von Paraffinschnitten gefriergetrockneten Gewebes; auf eine Rehydrierung in der feuchten Kammer kann im Gegensatz zur Verwendung von Paraffinschnitten gefriergetrockneten Gewebes verzichtet werden. — Für histologisehe Untersuchungen und Mucopolysaccharid-Nachweise gibt das Aufziehen der Schnitte in reinem Methanol nach vorheriger Bedampfung mit Formaldehyd (60 min, 20° C) die besten Ergebnisse. Für Enzymnachweise ist die Fixierung in Isopropylalkohol, für Dehydrogenasen in Aceton, am geeignetsten. Dabei gelingen der histochemische Nachweis der Cholinesterasen und der lysosomalen Enzyme besser als am konventionell behandelten Kryostatschnitt.

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The application of histological and histochemical techniques to freeze-dried cryostat sections

Summary The use of freeze-dried cryostat sections for various histological and histochemical techniques is described. It is shown, how sections can be mounted on slides and how they can be transferred into water-containing media. Following controlled freeze-drying artefacts due to watering are highly reduced as compared to paraffin sections of freeze-dried tissue; a re-hydration in a moist chamber is dispensable. — For histological purposes and investigations on mucopolysaccharides a formaldehyde vapour fixation (60 min, 20° C) followed by infiltration of the sections with pure methanol gives the best results. For enzyme histochemistry the postfixation with isopropanol is well suited, for dehydrogenase reactions acetone is recommended. — Histochemical reaction for cholinesterases and lysosomal enzymes on freeze-dried sections are superior to conventional techniques.

     

Penetration of stomata by liquids: dependence on surface tension, wettability, and stomatal morphology

Wettability of the leaf surface, surface tension of the liquid, and stomatal morphology control penetration of stomata by liquids. The critical surface tension of the lower leaf surface of Zebrina purpusii Brückn. was estimated to be 25 to 30 dyne cm⁻¹. Liquids having a surface tension less than 30 dyne cm⁻¹ gave zero contact angle on the leaf surface and infiltrated stomata spontaneously while liquids having a surface tension greater than 30 dyne cm⁻¹ did not wet the leaf surface and failed to infiltrate stomata. Considering stomata as conical capillaries, we were able to show that with liquids giving a finite contact angle, infiltration depended solely on the relationship between the magnitude of the contact angle and the wall angle of the aperture. Generally, spontaneous infiltration of stomata will take place when the contact angle is smaller than the wall angle of the aperture wall. The degree of stomatal opening (4, 6, 8, or 10 μm) was of little importance. Cuticular ledges present at the entrance to the outer vestibule and between the inner vestibule and substomatal chamber resulted in very small if not zero wall angles, and thus played a major role in excluding water from the intercellular space of leaves. We show why the degree of stomatal opening cannot be assessed by observing spontaneous infiltration of stomata by organic liquids of low surface tension.     

An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH

Summary A technique is described for highly sensitive and precise visualization of central catecholamine systems in paraffin sections of freeze-dried tissue. The procedure is based on perfusion of the animal with a solution containing formaldehyde and/or glyoxylic acid, in the presence of a very high magnesium content (40 g MgSO₄/150 ml solution) and acid pH. The perfused tissue is rapidly frozen, freeze-dried, treated with formaldehyde vapours (at +80°C for 1h), embedded in paraffin in vacuo, and finally sectioned.The present technique has a sensitivity for the dopamine- and noradrenaline-containing systems that is comparable with that of the glyoxylic acid-Vibratome technique, which utilizes fresh, glyoxylic acid-perfused tissue. Thus, the preterminal axon pathways become fluorescent throughout their full extent and the several new terminal systems, discovered with the glyoxylic acid-Vibratome method, are well demonstrable. The method is also highly useful for the study of the cell bodies and their dendritic processes. The catacholamine fibre systems are visualized without any signs of diffusion and with a richness in detail. In animals pretreated with l-tryptophan and MAO-inhibitor the technique is also useful for studies on central indolamine-containing systems.     

Methacrylate Embedding and Sectioning of Calcified Bone

Twenty-five milliliter aliquots of ethyl-butyl (1:1) methacrylate were polymerized at 6 or 7 initiator concentrations using 3 polymerization temperatures, both in air and in a water bath. Duplicate series were polymerized with and without vibration, pre-polymerization, and exclusion of oxygen. Hardening times and maximum temperatures reached within the samples were recorded. Vibration and the exclusion of oxygen had no effect. Prepolymerization, increasing polymerization temperature and increasing initiator concentration all decreased the hardening time and increased the maximum temperature. Polymerizing in a water bath rather than in air reduced the maximum temperature by 25-40°C and lengthened the hardening time about 1 hr. An initiator concentration of 0.4% Luperco CDB in ethyl-butyl methacrylate and a water-bath temperature of 45°C were selected for tissue embedding. The hardening time was 8 hr and the maximum temperature during polymerization was about 60°C.

Split rat femora and tibiae were freeze-dried and vacuum-infiltrated with acetone, absolute alcohol or monomer. The acetone or alcohol-fixed specimens were subsequently infiltrated with monomer. The specimens were transferred to 1 oz bottles, prepolymerized syrup added, and polymerized. No consistent differences between specimens treated by these methods were noted. Five-micron serial sections could be cut using a Leitz sledge microtome with a modified knife if the block was coated with paraffin between sections.     

Cellular Localization of CO(2) Fixation and Translocation of Metabolites

Leaves of maize (Zea mays L.) and sugar beet (Beta vulgaris L.) were enclosed in an illuminated chamber in air for 30 min after which time ¹⁴CO₂ was released into the chamber. Two min after the ¹⁴CO₂ was released, the leaves were removed from the chamber, and small sections were cut from them. The sections were put in small wire baskets and frozen in isopentane cooled by liquid nitrogen. Approximately 1.5 min elapsed from the removal of the leaf from the illuminated chamber until the tissue was frozen. The tissue was freeze-dried, embedded in paraffin and the cellular location of the isotopic activity was determined by radiography of leaf cross sections. Isotopic activity in maize leaves was localized in bundle sheath parenchyma. In contrast, the label in sugar beet leaves was generally distributed in the mesophyll cells. The bundle sheath cells in maize contain specialized chloroplasts which appear to have a unique capacity to incorporate CO₂. Translocation from leaves of maize was 3-fold as rapid as from sugar beet leaves in the same environment. Low light intensity did not alter the distribution pattern of fixed CO₂.     

Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy

Summary Cultured HeLa cells or mouse liver and pancreas tissues were labeled with ³H-thymidine, -uridine or -glycine for varying periods in vitro, frozen in liquid nitrogen and cut on an LKB ultrotome equipped with LKB Cryokit. Dry ultrathin sections were mounted on grid meshes and were either air-dried, freeze-substituted or freeze-dried, and were covered with dry films of radioautographic emulsions, exposed, developed, stained and were observed in electron microscopes.After a number of trials, it was possible to obtain fairly good preservation of both cell structure and radioisotopes by means of freeze-dried and drymounted ultrathin frozen sections. However, the results are not completely satisfactory at the present time.The outline of this paper was presented at the Symposium on Radioautography at the 5th International Congress of Histochemistry and Cytochemistry held in Bucharest, Romania, 30 August–2 September 1976     

A new method for demonstrating argyrophil cells of the pancreas and intestines

A new method for demonstrating argyrophil cells of the pancreas and intestinal tract using a combined silver and reducing solution in sections of formaldehyde fixed tissue is described. Impregnating sections in a 60 C water bath, the procedure takes about 25 min. A microwave version that takes about 5 min is also given. Results are similar to those obtained with the Grimelius method for argyrophil cells.     

A New Method for Demonstrating Argyrophil Cells of the Pancreas and Intestines

A new method for demonstrating argyrophil cells of the pancreas and intestinal tract using a combined silver and reducing solution in sections of formaldehyde fixed tissue is described. Impregnating sections in a 60 C water bath, the procedure takes about 25 min. A microwave version that takes about 5 min is also given. Results are similar to those obtained with the Grimelius method for argyrophil cells.     

Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15⁺) cells were investigated. A⁵¹Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15⁺ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15^–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with¹²⁵I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×10⁷ M^–1 and 2.8×10⁶ antigenic sites/cell.¹²⁵I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15⁺ cells.     

An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH.]

A technique is described for highly sensitive and precise visualization of central catecholamine systems in paraffin sections of freeze-dried tissue. The procedure is based on perfusion of the animal with a solution containing formaldehyde and/or glyoxylic acid, in the presence of a very high magnesium content (40 g MgSO4/150 ml solution) and acid pH. The perfused tissue is rapidly frozen, freeze-dried, treated with formaldehyde vapours (at +80 degrees C for 1 h), embedded in parffin in vacuo, and finally sectioned. The present technique has a sensitivity for the dopamine- and noradrenaline-containing systems that is comparable with that of the glyoxylic acid-Vibratome technique, which utilizes fresh, glyoxylic acid-perfused tissue. Thus, the preterminal axon pathways become fluorescent throughout their full extent and the several new terminal systems, discovered with the glyoxylic acid-Vibratome method, are well demonstrable. The method is also highly useful for the study of the cell bodies and their dendritic processes. The catacholamine fibre systems are visualized without any signs of diffusion and with a richness in detail. In animals pretreated with L-tryptophan and MAO-inhibitor the technique is also useful for studies on central indolamine-containing systems.     

Pituitary Secretory Granule Topography: Influence of Different Electron Microscopic Preparation Procedures on the Surface of Epon Sections

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO₄. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.     

Notes of Technic

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Beziehungen zwischen Polyaminen und Nucleinsäuren

Zusammenfassung Ausgehend von der Beobachtung, daß sich die Extrahierbarkeit von Putrescin und seiner Metabolite nach dem Exponieren von Kryostatschnitten in der sich bei 80° C über Paraformaldehyd ausbildenden Atmosphäre wesentlich verringert, wurde der Versuch unternommen, die unter Formaldehyd-Einfluß eintretende Fixierung der Polyamine zum Studium der intrazellulären Verteilung speziell von Putrescin und Spermidin zu nutzen. Die Fraktionierung von Lebergewebe in Lipide, Proteine und Nucleinsäuren ergab eine erhebliche Anreicherung von Spermidin in der Nucleinsäurenfraktion. Unter Berücksichtigung der Ergebnisse von Modellversuchen sind unsere Beobachtungen als ein Hinweis für die bevorzugte Lokalisation von Spermidin in nucleinsäurenhaltigen Strukturen der intakten Zelle zu deuten.

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Interrelationships between polyamines and nucleic acidsVII. Investigation of the intracellular localisation of putrescine and spermidine in freeze-dried and formaldehyde fixed cryostat sections

Summary Exposition of freeze-dried cryostat sections of tissue to formaldehyde vapour caused the binding of the polyamines spermine and spermidine, so that they were not extractable with acids. In persecution of this observation we isolated the protein, nucleic acid and lipid fraction of liver after the injection of radioactive putrescine to mice, at a time when radioactivity was mostly accumulated in spermidine. Though in model experiments the binding of the amines was most extensive to protein, we observed the most pronounced binding of radioactive spermidine to the nucleic acid fraction of the liver. This indicates, that spermidine is preferentially localised in vivo in nucleic acid containing structures.

     

Adherence of emulsan-producing Acinetobacter calcoaceticus to hydrophobic liquids

Summary The adherence of Acinetobacter calcoaceticus ATCC 31012 cells to hexadecane and perfluorocarbon FC-43 was measured using the Bacterial Adherence To Hydrocarbon (BATH) assay. In batch culture the adherence of cells to both hydrophobic liquids increased sharply during the exponential growth phase and remained high for the remainder of the culture period. No correlation was found between the surface emulsan concentration and the adherence to perfluorocarbon FC-43 and hexadecane. In continuous cultures, the production of cell-free emulsan was found to be growth-associated. The adherence to both hydrophobic liquids decreased with increasing dilution rate while the amount of surface emulsan increased. Furthermore, exogenously added emulsan decreased the adherence to hydrophobic liquids. Thus, the accumulation of surface emulsan does not appear to have a beneficial effect for cell adherence to hydrophobic liquids.     

Differential lytic and agglutinating activity of the anti-Lewisx monoclonal antibody FC-2.15 on human polymorphonuclear neutrophils and MCF-7 breast tumor cells. In vitro and ex vivo studies

The Lewis^x (Le^x) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le^x and has been previously shown to mediate the in vitro lysis of Le^x(+) cells by human complement. In a phase I clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In order to characterize FC-2.15 further and determine the physiological relevance of Le^x binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard analysis demonstrated an affinity constant of 5.14 × 10⁹ M⁻¹ and 1.11 × 10⁶ antigen sites/cell. In vitro, the binding of Le^x epitopes by FC-2.15 induced PMN homotypic aggregation, only 28.4 ± 4.1% remaining as single cells. When PMN and the Le^x(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In ⁵¹Cr-release assays employing human complement, FC-2.15 lysed 93.4 ± 7.9% of PMN and 87.8 ± 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pre-treatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 ± 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when ⁵¹Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking of Le^x epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le^x(+) tumor cells in circulation. Received: 3 December 1998 / Accepted: 28 January 1999     

Preservation of tissue mucins by freeze-drying and vapour fixation

Summary Histochemical studies previously undertaken showed that tissue mucins (glycoproteins) of the rat submaxillary salivary gland and other organs are preserved very satisfactorily by formaldehyde vapour treatment applied after freeze-drying of the tissue.It was thought desirable to confirm these histochemical findings by quantitative chemical data. This was performed by studying the effect of formaldehyde vapour treatment on the solubility of proteins in the freeze-dried rat submaxillary gland.Large quantities of protein (about 30 to 60 per cent of the dry weight) could be removed by aqueous extraction from the freeze-dried control samples, which had not received any formaldehyde vapour treatment, but very little protein (about 0.5 to 4 per cent of the dry weight) could be extracted from those samples which had been exposed to this vapour at 50°C for 3 hours.Each of the experiments performed confirmed this overall picture, but there were differences in the amount of protein extracted among the control samples, as well as among the formaldehyde vapour treated ones; it has been suggested that these differences were due to variations in the proportion and/or type of protein present, most probably caused by fluctuations in the content of secretory mucins.In part fulfilment for the Doctorate of Philosophy, University of London.     

Fusicoccin receptors. Evidence for endogenous ligand

The binding of fusicoccin to the microsomal preparations of maize roots in vitro is increased several-fold when segments of the tissue are washed for 2 h in distilled water before homogenization. Addition of freeze-dried wash solution to microsomal preparations of spinach leaves or fresh roots, washed roots, or coleoptiles of maize inhibited the binding of fusicoccin to particulate fractions. The freeze-dried material also blocked fusicoccin-promoted H⁺ extrusion from maize root segments. Roots may contain one or more water-soluble compounds competing with fusicoccin at the receptor level; such ligands might play a physiological role as modulators of the H⁺/K⁺ exchange system in higher plants.Abbreviation FC Fusicoccin     

Hyperchromicity and Strand Separation in Bacterial DNA

Studies of the per cent of strand separation of N¹⁴-N¹⁵ hybrid coli DNA heated to various temperatures in formaldehyde have shown that the process of strand separation is a function of temperature and formaldehyde concentration and is directly related to the measured hyperchromicity. No strands separate until about 75 per cent of full hyperchromicity is obtained, and even at apparently full hyperchromicity a large fraction of the strands may be held together, possibly by guanine-cytosine-rich regions.