Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

山茶属花粉外壁表面微形态特征的研究

在前人研究的基础上,利用光学显微镜和扫描电镜观察了山茶属17个组34个代表种（含变种）的花粉形态;按照韦仲新划分山茶属花粉类型的标准,对其进行归类,所有花粉分为3类：颗粒状至皱颗粒状纹饰、皱沟状纹饰和穴－网状纹饰;发现1种新的花粉类型：拟穴－网状纹饰。本文还对山茶属的某些分类学问题作了初步探讨。     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue

Summary Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5–1%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.     

Light microscopic identification and photography of Golgi impregnated central nervous system neurons during sectioning for electron microscopy.

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin sections of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which part of the neuron and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Light Microscopic Identification and Photography of Golgi Impregnated Central Nervous System Neurons During Sectioning for Electron Microscopy

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin door of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which put of the and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Ultrastructural observations on the biogenic amines in the carotid and aortic-abdominal bodies of the human fetus

Summary The carotid and aortic-abdominal bodies of two human fetus at fifth month of pregnancy have been studied with the light and electron microscope. A personal variation of the glutaraldehyde ammoniacal silver (GA/S) method has been used, which consists in performing the silver reaction on the ultramicrotomical sections of the tissues, first fixed by glutaraldehyde perfusion and then included in Epikote 812.In the light microscope, the GA/S reaction is certainly positive in the aortic-abdominal bodies and it is negative or dubious in the carotid bodies. — In the electron microscope, however, the reaction is positive both in the aortic-abdominal and in the carotid bodies. — In the aortic-abdominal bodies, the silver precipitate accumulates into thick cytoplasmic granules, which have been shown to be NA-storing granules. — In the carotid bodies however, the silver precipitate accumulates into much smaller cytoplasmic granules, which are interpreted as 5-HT-storing granules.     

THE DISTRIBUTION OF SYNAPSES ON A PHYSIOLOGICALLY IDENTIFIED MOTOR NEURON IN THE CENTRAL NERVOUS SYSTEM OF THE LEECH : An Electron Microscope Study after the Injection of the Fluorescent Dye Procion Yellow

The fine structure of a physiologically identified motor neuron in the segmental ganglion of the leech central nervous system and the morphology of synapses on it were studied after injection of the fluorescent dye Procion yellow as a marker. The injected cell and its processes within the neuropil were located in thick or thin sections with fluorescence optics after initial fixation with glutaraldehyde and brief treatment with osmium tetroxide. The same or adjacent thin sections could then be examined in the electron microscope. Comparison with uninjected cells showed that the general features of the injected cell are retained although some organelles are distorted. The main features of the geometry of this neuron are the same from animal to animal: a single large process runs from the soma through the neuropil to bifurcate and enter the contralateral roots. Within the neuropil the main process gives off long branches (up to 150 µ), but these are greatly outnumbered by short branches and spines, one or a few microns in length, which were not appreciated in previous light microscope studies after injection of Procion yellow. Serial thin sections of selected areas along the main process within the neuropil showed that there are synapses on most of the shorter branches and spines; occasional synaptic contacts were also made on the main process itself and on longer branches. At least two morphologically distinct types of synapse could be recognized. A minimum estimate of the total number of synapses on the motor cell is 300, based on their occurrence in reconstructed segments.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB- and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.     

Simultaneous characterization of efferent and afferent connectivity, neuroactive substances, and morphology of neurons.

We present a method for establishing in a single experiment four characteristics of individual neurons: the efferent and afferent connectivity, the morphology, and the content of a particular neuroactive substance. The connectivity of the neurons is determined by retrograde fluorescent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-micron thick slices. Neurons containing retrogradely transported Fast Blue are intracellularly injected with the fluorescent dye Lucifer Yellow to fill their dendritic trees. The slices are then resectioned at 20-40 microns. One section through the soma of a Lucifer Yellow-filled neuron is selected for the detection of a neuroactive substance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filtering, it can be determined in the fluorescence microscope whether a Lucifer Yellow-containing cell body has also been labeled with TRITC, i.e., whether it is immunoreactive for this neuroactive substance. The adjacent sections are subjected to dual peroxidase immunocytochemistry with different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer Yellow (diaminobenzidine, brown reaction product) in the dendrites of the intracellular injected cells. The other sections are used for electron microscopic visualization of the transported PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow-injected, retrogradely Fast Blue-labeled cells (brown color) are studied by light microscopy. The electron microscope supplies ultrastructural data on the PHA-L-labeled axon terminals.     

Demonstration of Lipofuscin and Nissl Bodies in Crystal Violet Stained Sections Using a Fluorescence Technique or Pyronin Y Stain

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures—Nissl bodies and lipofuscin—can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

Summary The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB-and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.Supporied by grants from the Ministry of Education, Science and Culture of Japan     

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.     

Simultaneous fixation and production of catecholamine fluorescence in central nervous tissue by perfusion with aldehydes

Synopsis Perfusion with a mixture of formaldehyde (4%) and glutaraldehyde (0.5%) is shown both to fix central nervous tissue and to produce, with no further treatment, a fluorescence histochemical localization of catecholamines. After perfusion, serial sections can be readily taken through the whole brain with a Vibratome. Landmarks which are apparent at low power with white-light illumination can be seen when the sections are viewed in the fluorescence microscope. Catecholamine-containing nerve cell bodies, varicose axon terminals and non-varicose nerve fibres appear brightly fluorescent and well localized. The method has been applied to rats, guinea-pigs and rabbits and is ideally suited to the accurate mapping of central catecholamine neurons and their processes.     

Characterization of hypothalamic neurons expressing a neuropeptide receptor, GALR2, using combined in situ hybridization-immunohistochemistry.

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.