Combination of metallic impregnation and autoradiography of brain sections

Summary After labelling with ¹⁴C-thymidine, frozen sections or paraffin sections of the brain of adult mice or rats were first stained by metallic impregnation and then coated with chrome alum gelatine and with an emulsion layer of about 10 m. On the autoradiographs ¹⁴C-tracks are readily recognized above labelled astrocytes or oligodendrocytes, and these can be well discriminated, if the sections are processed by the silver carbonate method of Rio-Hortega. In contrast, no labelling is obtained, if the gold chloride sublimate method of Cajal is applied.     

High-resolution in situ hybridization and TUNEL staining with free-floating brain sections.

We applied in situ hybridization and the TUNEL technique to free-floating (vibratomed) sections of embryonic and postnatal mouse CNS. Full-length cDNAs specific for oligodendrocyte- or astrocyte-specific genes were labeled with digoxigenin using the random primer method. With paraformaldehyde-fixed sections, the nonradioactive in situ hybridization method provides detection of individual, very small glial progenitor cells in embryonic development. Small, isolated cells expressing oligodendrocyte specific messages can be detected in the neuroepithelium at embryonic and postnatal stages. The technique can be completed within 3 days and is as sensitive as the radioactive method. Likewise, the TUNEL method using DAB as the chromogen on free-floating sections provides excellent resolution. These DAB-stained sections can be embedded in plastic and thin-sectioned to visualize the ultrastructure of apoptotic cells. Both in situ hybridization and TUNEL methods can be applied to the same section, the tissue embedded in plastic, and semithin sections cut. The high resolution obtained with this combined procedure makes it possible to determine whether brain cells expressing glia-specific messages are undergoing apoptosis.     

Further data on the development of SRIF-like immunoreactive nerve cell populations in the chick embryo brain stem. I. Medulla and pons.

Further immunocytochemical analysis of the neuroblasts with SRIF-like immunoreactivity (ir) was carried out on the chick embryo medulla and pons. 5 or 100 microns rombencephalon sections were obtained from 60 White Leghorn chick embryos at stages (E = Embryonic days) ranging from E4 1/2 to E18 and incubated with rabbit polyclonal antibodies against synthetic cyclic Somatostatin-14, according to PAP-DAB technique. In the medulla and pons the ir appeared as from E12. From E12 to E13 1/2-E14 the ir distribution gradually changed. From E14 to E18 numbers and spatial arrangement of the positive neuroblast groups did not show substantial changes; in these respects the ir distributional pattern proved to be markedly different from the one observed by the Authors in adult animals. Moreover, from E13 to E15 the positive neuroblast density appeared to be higher than that of positive neurons in adults. These results are consistent with a possible SRIF local regulative role.     

Further data on the development of SRIF-like immunoreactive nerve cell populations in the chick embryo brain stem. II. Midbrain tegmentum.

Further immunocytochemical analysis of the neuroblasts with SRIF-like immunoreactivity (ir) was carried out on the chick embryo midbrain tegmentum. 5 or 100 microns mesencephalon sections were obtained from 60 White Leghorn chick embryos at stages (E = Embryonic days) ranging from E4 1/2 to E18 and incubated with rabbit polyclonal antibodies against synthetic cyclic Somatostatin-14, according to PAP-DAB technique. In the midbrain tegmentum the ir appeared as from E12. From E12 to E13 1/2-E14 the ir distribution gradually changed. From E14 to E18 numbers and spatial arrangement of the positive neuroblast groups did not show substantial changes; in these respects the ir distributional pattern proved to be similar to the one observed by the Authors in adult animals. From E17 to E18 a decrease in the positive neuroblast density appeared to occur, particularly in a ventrally placed group. These results are consistent with a possible local regulative role of the SRIF.     

High-resolution digital brain atlases: a Hubble telescope for the brain

We describe implementation of a method for digitizing at microscopic resolution brain tissue sections containing normal and experimental data and for making the content readily accessible online. Web-accessible brain atlases and virtual microscopes for online examination can be developed using existing computer and internet technologies. Resulting databases, made up of hierarchically organized, multiresolution images, enable rapid, seamless navigation through the vast image datasets generated by high-resolution scanning. Tools for visualization and annotation of virtual microscope slides enable remote and universal data sharing. Interactive visualization of a complete series of brain sections digitized at subneuronal levels of resolution offers fine grain and large-scale localization and quantification of many aspects of neural organization and structure. The method is straightforward and replicable; it can increase accessibility and facilitate sharing of neuroanatomical data. It provides an opportunity for capturing and preserving irreplaceable, archival neurohistological collections and making them available to all scientists in perpetuity, if resources could be obtained from hitherto uninterested agencies of scientific support.     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

Loading of assimilates in wheat leaves. II. The path from chloroplast to vein

Abstract. An improved method is described for micro-autoradiography of water-soluble substances alter freeze-substitution of plant tissue in which water is rigorously excluded. Resin sections are cut and flattened dry, and dry photographic emulsion is mounted on them. When the location of ¹⁴C in wheat leaves after assimilation of ¹⁴CO₂ was studied with this method, it was found that ¹⁴C entered the intermediate veins before the laterals and entered both types of veins along the flanks of the veins adjacent to the phloem. High concentrations of ¹⁴C were found in small spaces in the cells of mesophyll and vein parenchyma; these spaces coincide with the nuclei. The concentration of ¹⁴C in these nucleus-associated spaces was as high as that reached at later times in the sieve tubes. Water washed the ¹⁴C out of these spaces of the sections and the label in the washings was predominantly in sucrose. The high ¹⁴C concentrations of the nucleus-associated spaces were particularly easily leached. It is concluded that the raising of the sugar concentration to the high levels found in sieve tubes can take place in these leaves in a special space in each mesophyll cell not, or not only, at the boundary of the sieve tubes.     

Selective adherence of lymphocytes to myelinated areas of rat brain.

An in vitro system developed for studying lymphocyte binding to high endothelial venules (HEV) of lymph nodes was used to determine if there are similar binding sites in other organs of the rat. Thoracic duct lymphocytes (TDL) adhered selectively and uniformly to white matter when overlaid onto glutaraldehyde-fixed tissue sections of cerebellum and cerebrum. The pattern of TDL adherence to cerebellar sections showed that binding to nonmyelinated areas was negligible. Comparison of TDL-white matter to TDL-HEV binding demonstrated that the density of adherence to each site was quantitatively similar. In contrast, lymphocytes exhibited little tendency to bind to tissue sections of liver, spleen, heart, thymus, and salivary glands. TDL adherence to cerebellar white matter occurred rapidly, was cell dose dependent and optimal at 7 degrees C. White matter binding was also a property of spleen lymphocytes but the thymus was deficient in cells with this capability. The affinity of TDL and spleen lymphocytes for myelinated areas of the brain suggests the presence of myelin binding receptors on these cells.     

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Immunohistochemical labeling of androgen receptors in the brain of rat and monkey.

Androgen receptor antibodies have recently been developed using fusion proteins containing fragments of human prostatic androgen receptor. We have used a polyclonal antibody raised in rabbits to label androgen receptors in brain sections from male and female rats and monkeys. Free-floating frozen sections were incubated in primary antibody, and processed by the peroxidase-avidin-biotin complex method using biotinylated anti-rabbit IgG. Nickel intensified diaminobenzidine was used as the chromagen, and neurons were labeled in the amygdala, hippocampus, bed nucleus of stria terminalis, septum, preoptic area, in several hypothalamic nuclei including the supraoptic and paraventricular nuclei, in several brain stem motor nuclei and in cerebral cortex. Staining was most intense in cell nuclei but also occurred in cytoplasm and in some neuronal processes. Labeling was more restricted in monkey than in rat brain. Omitting the primary antibody or pre-incubating the primary antibody with rat prostatic cytosol for control purposes demonstrated the specificity of staining.     

Independent brain stem sites for ventilatory neurogenesis

The purpose was to determine if independent ventilatory rhythms could be generated in each half of completely separated brain stems. In decerebrate, cerebellectomized, vagotomized, paralyzed, and ventilated cats, activities of phrenic, recurrent laryngeal (RLN), and/or hypoglossal nerves were monitored. Midsaggital brain stem divisions were performed by sections and lesions. Eupnea continued following divisions of mesencephalon and pons. Hypoglossal and phrenic activities were eliminated after sections approximating the obex. In most preparations, RLNs discharged with independent rhythms after completion of midsagittal brain stem section and a C1 transection. Independent rhythms were also obtained from each half of medulla following transections at the pontomedullary junction and at C1, and midsagittal medullary divisions. In other animals with transections between pons and medulla and at C1, synchronized RLN and hypoglossal activities persisted after sagittal medullary divisions, 2.0 mm lateral to midline contralaterally. Data demonstrate that there is more than one potential brain stem site for ventilatory neurogenesis. It is hypothesized that there are many such sites, possibly having pacemaker cells, in pons and medulla.     

Plastination of dissected brain specimens and Mulligan-stained sections of the human brain

The difficulties in obtaining human brain material for teaching neuroanatomy have increased the demand for more durable brain specimens. In this paper, we describe results obtained by preparing large, plastinated, dissected human brain specimens and Mulligan-stained sections of the human brain. The brains were fixed in formalin, washed and dissected in order to visualize the fibre tracts and larger nuclei in the central nervous system. This was followed by dehydration at -20 degrees C in acetone. The specimens were then impregnated with silicone, Biodur S10, in vacuo and hardened in Biodur S6 vapour. The grey and white substance in the central nervous system as well as the larger fibre tracts and nuclei were clearly visible in the dissected, plastinated specimens. Coronal and sagittal sections of the human brain were stained according to Tompsett's modification of the Mulligan method. The sections were then dehydrated in cold acetone followed by forced impregnation with Biodur S10 and hardening. The plastinated sections stained distinctly and strongly and the nuclei in the forebrain, cerebellum and brain stem could be identified easily. The sections did not fade when exposed to light and could be easily handled in the classroom without damage. Therefore, the distinct visualization of neuroanatomical structures, the improved durability of the specimens, as well as the lack of odour make plastinated specimens and stained sections of the central nervous system a valuable tool for teaching neuroanatomy that compliments the use of wet preparations.     

Histochemical Use Of Lead Tetra-Acetate I. Cleavage Of 1-2 Glycols

Lead tetra-acetate acts specifically to split the carbon-carbon single bond of the 1,2-glycol linkage to produce aldehyde radicals which may then be demonstrated by means of leucofuchsin, 2,4-dinitrophenlyhydrazine, or p-nitrophenylhydrazine. Routinely prepared slide sections from tissues fixed in 10% formalin are run down to 95% alcohol, rinsed in glacial acetic acid and then treated for 2 minutes in a saturated solution of lead tetra-acetate in glacial acetic acid with 5 g. of potassium acetate added for each 100 ml. of reagent. The sections are then washed in distilled water and placed in leucofuchsin for 10 minutes, or in a saturated 30% alcoholic solution of p-nitrophenylhydrazine for 5 minutes or 2,4-dini-trophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 minutes if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofuchsin or yellow by the nitrophenylhydrazines.     

Biosynthesis of avenacins and phytosterols in roots of Avena sativa cv. Image

In keeping with the proposal that avenacin biosynthesis is restricted to the tips of primary roots of oat seedlings, the incorporation of radioactivity from R-[2-(14)C]mevalonic acid (MVA) into avenacins and beta-amyrin by serial sections of primary roots was found to be more-or-less restricted to root tip sections. Squalene synthase (SQS) (EC 2.5.1.21) and 2,3-oxidosqualene:beta-amyrin cyclase (OS beta AC) (EC 5.4.99) were also most active in these sections. The incorporation of radiolabel from R-[2-(14)C]MVA into cycloartenol and 24-methylene cycloartanol by, and the 2,3-oxidosqualene:cycloartenol cyclase (OSCC) (EC 5.4.99) activity in, the various serial sections were consistent with phytosterol biosynthesis occurring in all the sections of the root with some tailing-off in the rate of synthesis in the more distal sections.     

Tannic acid-iron alum reactions: stain of choice for macroscopic sections of brain to be embedded in plastic.

As a macroscopic stain for gross brain sections to be embedded in plastic, tannic acid-iron alum is superior to the generally recommended LeMasurier's variation of the Berlin blue technique because of its greater permanency in plastic. However, as originally adopted for use with brain tissue by Mulligan, the intense black staining of gray matter is too dark for plastic embedded specimens. A modification of this method designed to overcome this difficulty is described. Staining procedure: Wash formalin-fixed brain slices overnight in running water. Wash in distilled water, 2 changes, 30 minutes each. Place slices individually in Mulligan's solution at a temperature of 60-65 C for 4 minutes. Rinse in ice water for 10 seconds. Mordant in 0.4% tannic acid in distilled water for 1 minute. Wash in running tap water for 1 minute. Develop in 0.08% ferric ammonium sulfate in distilled water until gray matter is light gray, about 10-15 seconds. Wash in lukewarm running water for 1 hour, then gently hand-rub whitish film from myelinated surfaces. Store briefly in 3% formalin or 25% glycerine if necessary depending on plastic embedding procedure to be followed.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

Fluorescent detection of APUD-type cells in the digestive tract of the rabbit fetus. Correlative study in electron microscopy

Previous studies performed on different species have shown that these cells could be recognized by their morphologic and immuno-histological features. In early stages, these cells are able to take up and decarboxylate amine precursors. Therefore the aim of the present work was to determine if this uptake could be correlated with ultrastructural modifications. A processing technique allowing amine detection and correlative ultrastructural examination was used. Rabbit foetuses 13, 14, 17 and 21 day old were studied. The gastro-intestinal tracts of L-DOPA treated or untreated foetuses were removed in a glutaraldehyde-formaldehyde mixture and embedded in epoxy-resin. Semi-thin sections allowed to locate fluorescent cells in U.V light microscopy; adjacent thin sections were observed in electron microscopy. The first green fluorescent cells appeared in the 13 day old foetuses treated with L-DOPA. By this stage, these cells were very scarce and appeared poorly differentiated in electron microscopy. Between the 15th and the 18th day, the green fluorescent cells contained only small round granules. By the day 19, orange-yellow cells can be observed in L-DOPA treated and untreated foetuses. These cells possessed characteristic enterochromaffin granules. The green fluorescent cells of 21 day old foetuses, treated with L-DOPA, exhibited various fluorescence intensities correlated with the heterogeneity of the secretory granules. Some foetuses of each stage were treated with Falck's technique. This method gave similar results concerning the chronology of fluorescent cell detection.     

Gallocyanin as A Nuclear Stain

Gallocyanin has been used successfully as a nuclear stain. Sections are cut by the freezing method of either fixed or unfixed tissue. The tissues are warmed (not exceeding 70°C.) for 2-4 minutes in the gallocyanin solution. A counterstain may be used if desired. The most effective are Biebrich scarlet, phloxine, or eosin Y. The sections are then dehydrated and mounted in clarite. The nuclear pattern is clearly demonstrated and the sections are permanent.