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.     

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.     

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 [¹⁴Clproline 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 wholebody 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. Wholebody and light microscopic antoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in wholebody sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.     

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.     

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.     

Tracing neuronal connections with radioisotopes applied extracellularly.

Methodological and technical problems of the autoradiographic-neuroanatomical tracing (ARNT) technique are discussed. The size of the labeled area after a tritiated amino acid injection varies directly with the volume of isotope, the rate of injection, and the length of exposure of the secretion to the emulsion. Frozen sections can be used for autoradiography if they are mounted on subbed slides, dehydrated in ethanol, defatted for 1 hour in xylene, rehydrated through ethanol and water, and dried before coating with emulsion. Brushes used for mounting frozen sections should be used only for this purpose and dipped in boiling distilled water before use to avoid chemoreduced streaks in the emulsion due to contamination from the brushes. Excessive dilution of Kodak emulsion can leave less than a monolayer of grains over certain types of sections; the emulsion thickness should be checked by exposing coated test sections to a brief flash of light and developing immediately. A high intensity safelight recommended for the ARNT darkroom is the Thomas Duplex Super monochromatic sodium vapor bulb safelight; for Kodak NTB-2, -3 and Ilford L4 emulsions the red-banded and yellow-banded filters are used. A useful stain combination for ARNT is Luxol fast blue stained before coating and cresyl violet stained after developing which demonstrates both neuronal cell bodies and myelinated tracts in the same section.     

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.     

A Rapid Method for Resectioning 0.5-4 μ Epoxy Sections for Electron Microscopy

A simple and rapid method is described for resectioning semithin Epon sections which have been stained for light microscopy, mounted on slides, and examined under immersion oil. The immersion oil is removed with xylene and the section is air dried. A drop of distilled water is applied to the slide and a razor blade is slid under the section. Freed from the slide, the section floats on the surface of the water and is transferred to another drop of water on the surface of a smooth, newly prepared Epon block face. The water under the section is withdrawn with bibulous paper. The section is thoroughly dried and bonded to the block surface by briefly heating in a 60 C oven. The tissue may then be re-sectioned and stained for electron microscopy in the conventional manner. This method has been used by several different technicians to produce ultrathin sections equal in quality to those produced by conventional methods and it greatly facilitates the selection of critical areas for examination by electron microscopy.     

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.     

Differentiation of Gram Positive and Gram Negative Bacteria in Transparent Acrylic Resin Emulsion Replicas of Surfaces of Plants

When acrylic resin emulsion (Rohm & Haas Primal AC-33) is allowed to dry on a leaf surface, it can be peeled off to give a transparent replica of the surface with bacteria and fungi embedded in it. The distribution of micro-organisms in the replica appears to reflect the patterns in which they occur naturally on the surface. The resin replicas may be stained by a variety of microscopical stains, the best of which is phenol-acetic-aniline blue, and are suitable for high power light microscopy.
Acrylic resin emulsion differs from widely used cellulose-based materials in that permanent differentiated preparations of Gram positive and Gram negative stained bacteria may be produced from a wide variety of types of surface.     

A Method to Prevent Wrinkles in Autoradiographic Emulsion when Staining Plastic Embedded Sections with Hematoxylin and Eosin-Phloxine

A procedure was developed which prevents wrinkles in autoradiographic emulsion when sections, embedded in glycol methacrylate, are stained with hematoxylin and eosin-phloxine. Craniofacial tissues labeled with tritiated thymidine were collected and mounted on slides. Slides were dipped in emulsion, stored for one month and developed. The slides were immersed in liquefied celloidin and subsequently stained with a modified hematoxylin and eosin-phloxine procedure. Results showed that the emulsion did not wrinkle and the procedure did not effect the occurrence of labeled cells.     

Identity of Holmes Positive Bodies with Electron Microscopically Demonstrable Nucleolus-Like Bodies in Neuronal Cytoplasm

By light microscopic observation of mouse brain stained by Holmes' silver method deeply stained cytoplasmic inclusion bodies were seen in almost all nerve cells of the locus coeruleus. Electron microscopy of tissue samples from floating Vibratome sections stained by Holmes' silver method demonstrated that the nucleolus-like bodies in the cytoplasm were densely impregnated with gold particles. Hence, it was confirmed that the cytoplasmic inclusion bodies of paraffin sections stained by Holmes' method are identical to the so-called nucleolus-like bodies seen in electron microscopic studies.     

Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Radioautography of Histologic Sections of Human Testicular Tissue

Two techniques suitable for qualitative radioautographic analysis of histologic sections of human testicular tissue are described. In one, deparaffinized wet slides are coated with photographic emulsion resulting in significant improvement in preservation of histologic quality of the tissue section. In the second procedure stained and processed slides are coated with a thin layer of H.S.R. prior to coating with photographic emulsion. This procedure allows excellent preservation of the stained tissue section and also enables visualization of the reactive cell and its radioautographic image at two different focal planes. The techniques can be applied with equal advantage to histologic preparations of other tissues.     

Picrosirius Red Staining of Dental Structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Picrosirius red staining of dental structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.