A method for preparing whole-body sections suitable for autoradiographic, histological and histochemical studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [14C]proline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For whole-body autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Whole-body and light microscopic autoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in whole-body sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.     

Light microscopic autoradiography for study of early changes in the distribution of water-soluble materials

An approach using autoradiography for the study of early changes in the distribution of water-soluble materials and the chemography involved was investigated. Radioactive calcium chloride (45Ca) was injected into the iliac vein of a rat. Ten seconds after the injection the rat was frozen in hexane (-90 degrees C). The frozen rat was embedded in 5% sodium carboxymethyl cellulose and blocked in the coolant. A sheet of plastic tape coated with a synthetic rubber glue was fastened to the trimmed block surface, and whole-body sections 2-10 microns thick were cut with a disposable microtome knife. Selected sections were freeze-dried and then covered with a dried autoradiographic emulsion film about 1 microns thick. The autoradiograph clearly showed the distribution of radioactive calcium in the calcification zone of long bones. The samples chosen to assess chemographic artifacts showed positive and negative chemographies on most of the tissues when these were kept at 23 degrees C, and although both chemographic effects were significantly reduced when the samples were kept at -20 degrees C, cells in several tissues still exhibited positive and negative chemographies. The technique can be used for the study of any animal whose size is suitable for whole-body freeze-sectioning.     

An innovative coating technique for light and electron microscopic autoradiography.

We describe a modified nuclear emulsion coating technique for both electron and light microscopic autoradiography. We propose that by reversing the application of formvar film so that it adheres to and covers thin sections placed on grids, we have developed a technically accessible methodology that produces optimal conditions for the tracing of specific nuclear activity. A smooth, continuous base is formed over the sections on which a monolayer of evenly packed silver halide crystals can be applied by dip-coating. The same principle is applied to pre-stained 1-micron plastic sections of glass slides. We suggest that the application of formvar film over thin sections does not impede or interfere with the exposure of the emulsion by the labeled tissue. On the contrary, it virtually eliminates contamination and background radiation, enhancing the specificity and quality of resolution at even low magnifications. This technical modification, which facilitates the application of the emulsion, could render electron microscopic autoradiography a routine laboratory procedure, allowing for easily reproducible results and quantitative evaluation. At the light microscopic level, this technique prevents chemical fogging caused by certain stains, and thus allows routine pre-staining before coating with emulsion.     

Dry, High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

Dry,High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

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.

     

Prevention of Chemography in Prestained Epoxy Autoradiographs by an Intervening Carbon Film

Evaporation of a carbon layer over toluidine blue stained epoxy sections prior to applying emulsion eliminates or significantly reduces chemography that would otherwise be present in autoradiographs. This simple procedure permits routine proceasing of large numbers of slides without the limitations of the impermeable membranes currently recammended for light microscopic autoradiography following prestaining. The method permits “before and after” microphotography and use of simple staining procedures for sections to be studied autoradiographically.     

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris.

A method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-micron sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried. Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue. To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.     

Tannic acid-metal salt sequences for light and electron microscopic localization of complex carbohydrates.

Use of tannic acid (TA), in sequence with ferric chloride, uranyl acetate or gold chloride resulted in staining of selective but sometimes different sites in paraffin sections. TA-uranyl acetate of TA-ferric chloride stained sites rich in complex carbohydrates, wherease TA-gold chloride stained the collagen of various connective tissues different shades of red-purple to gray-black. Applied to epoxy-embedded thin sections of tissues fixed with glutaraldehyde and not post-osmicated, TA-uranyl acetate and TA-ferric chloride imparted density to subcellular sites known to contain a high concentration of mucosubstances, such as secretory granules and cisternae of the Golgi complex of certain cells. TA-gold chloride proved unsatisfactory for ultracytochemistry because of its tendency to form globular precipitates on thin sections. The effect of blockage procedures at the light microscopic level indicated that vicinal glycols are not required for binding of TA to tissue sites. Electrostatic forces were shown to be of minimal significance, whereas hydrogen bonding appeared to play a part in both TA-tissue and TA-metal binding mechanisms.     

Prevention of negative chemography in phenylenediamine stained autoradiographs of plastic semithin sections

Negative chemography is the loss of latent image during autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with glutaraldehyde and osmium tetroxide, block stained with p-phenylenediamine and embedded in Epon for light microscope sections causes intense negative chemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the chemography. Treatment with 1% hydrogen peroxide for 15 min reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.     

Prevention of Negative Chemography in Phenylenediamine Stained Autoradiographs of Plastic Semithin Sections

Negative chemography is the loss of latent image daring autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with ghuaraldehyde and osmium tetroxide, block stained with ft-phenylenediamme and embedded in Epou for light microscope sections causes intense negative cbemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the cbemography. Treatment with 1 % hydrogen peroxide for 15 nun reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.     

Prevention of Chemography in Prestained Epoxy Autoradiographs by an Intervening Carbon Film

Evaporation of a carbon layer over toluidine blue stained epoxy sections prior to applying emulsion eliminates or significantly reduces chemography that would otherwise be present in autoradiographs. This simple procedure permits routine proceasing of large numbers of slides without the limitations of the impermeable membranes currently recammended for light microscopic autoradiography following prestaining. The method permits “before and after” microphotography and use of simple staining procedures for sections to be studied autoradiographically.     

A Sudan Black B Myelin Stain for Peripheral Nerves

Blocks of fresh issue were fixed 2 or more days in: cobalt sulfate (or nitrate), 1 gm; distilled water, 80 ml; 10% calcium chloride, 10 ml; and formalin, 10 ml. The fixed tissue was washed thoroughly in tap water, embedded in gelatin, frozen sections cut, and mounted on slides with gelatin adhesive. The sections were stained 15-30 min in a saturated, filtered solution of Sudan black B in 70% alcohol, differentiated in 50% alcohol under microscopic observation, and a cover glass applied with glycerol-gelatin. In thick (50-100 μ) sections, myelin stained green to gray-green and this allowed easy differentiation between nerves and other tissue elements.     

TiO2膜吸附固定糖化酶特性的研究

分别以醋酸纤维素TiO2膜(AC.TiO2膜)、羧甲基纤维TiO2膜(CMC.TiO2膜)和聚丙烯TiO2膜(PP.TiO2膜)为载体吸附固定糖化酶,并与醋酸纤维素、羧甲基纤维素和聚丙烯固定糖化酶的性能进行了比较,得出以AC.TiO2膜和PP.TiO2膜对糖化酶的吸附性能及稳定性能均较好,PP.TiO2膜固定的糖化酶使用8次后其剩余酶活仍能保持在72%.     

The use of slotted grids for electron microscopic radioautography

A method for electron microscopic radioautography on slotted grids is presented which allows examination of the distribution of silver grains over sections of entire structural units without interference by grid bars. Tissue sections of a size such as to fit the opening of the grid slot are placed on slides coated with a Formvar film of sufficient strength to permit transfer of the completed radioautograph onto the grid and to support it over the slot. Sections are block stained prior to radioautography to minimize the risk of loss of the radioautograph during the procedure.     

The Use of Slotted Grids for Electron Microscopic Radioautography

A method for electron microscopic radioautography on slotted grids is presented which allows examination of the distribution of silver grains over sections of entire structural units without interference by grid bars. Tissue sections of a size such as to fit the opening of the grid slot are placed on slides coated with a Formvar film of sufficient strength to permit transfer of the completed radioautograph onto the grid and to support it over the slot. Sections are block stained prior to radioautography to minimize the risk of loss of the radioautography during the procedure.     

An electron microscopic histochemical and X-ray microprobe study of spherites in a mussel

Transmission electron microscopic, histochemical and X-ray analytical microprobe techniques were used to study the inorganic-organic relationship in the spherites (calcospherules) from the mantle, i.e. subadjacent to the outer mantle epithelium, of the fresh-water mussel Amblema. These structures were shown to contain calcium which could be chelated by the flotation of sections on solutions of either formic acid or ethylene glycol bis-(beta-amino ethyl ether)-N, N1-tetra-acetic acid (EGTA), Analysis of both non-chelated and sections revealed a significant sulfur peak. Chelated spherites were also intensely stained with acid phosphotungstic acid (PTA), Such data is indicative of the presence of an organic glycoprotein (proteoglycan) matrix which could serve to bind mineral ions, thus forming organic-inorganic aggregates for calcium transport and homeostasis. In this regard, the spherites are analogous to both calcium phosphate containing mitochondrial granules and the initial calcification sites in vertebrate mineralizing tissues.     

Fluorescamine, a fluorescence probe for amino groups in histochemical studies of plant cells and the effect of mercury fixation

Synopsis When fixed in mercuric chloride solutions and stained with Fluorescamine, histological plant specimens emit a strong fluorescence. The fluorophore distribution is topologically identical to the staining pattern revealed by visible light methods for nucleoproteins, but the fluorescence mode of viewing preparations gave greater sensitivity and contrast than transmitted light absorption methods. The parameters that influence the formation of the fluorescent image in plant cells are discussed. The results obtained indicate that the mercury-Fluorescamine reaction is an ideal histochemical procedure for collecting qualitative and analytical information on plant nuclei and on the changes of nucleolar architecture that occur during the cellular developmental cycle.     

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris

Summary A method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-m sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried.Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue.To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.     

Staining method for whole-body autoradiography.

Sagittal whole-body sections of frozen mice were cut on a hydraulicly driven microtome in a cryostat at--15 C by applying cotton or nylon-backed adhesive tape to the mouse before cutting. Section thickness was 20 mu. The sections, still adhering to the tape, were dried in the cryostat (-15C) under atmospheric pressure. After autoradiography, the sections were pressed to a glass slide spread with a mixture of albumin and glycerin. The slide was immersed in xylene at 30 C for 15 min. The tape was then removed from the slide, where the section remained to be stained with hematoxylin-eosin. The section thus obtained enabled the tissue histology to be related to the autoradiogram. This method may also be applied to histochemical studies of substances insoluble in xylene.