A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

Spectrophotometric and cytochemical studies on azures A, B and C.

The cytochemical use of azures A, B and C, propared with either N HCL and potassium metabisulphite or with sodium hydrosulphite in tissue sections were investigated. Both in situ absorption curves of nuclei stained with each of these dye-SO2 reagents as well as in vitro absorption data of acqueous solutions of the dyes are also presented. It has been pointed out that the mechanism of staining with azure A-SO2 and azure C Eosinate-SO2 is the same as that of the conventional Feulgen reaction with Schiff reagent but that of staining with azure B-SO2 is by the modified Feulgen reaction because this dye does not contain any primary amino group.     

The Use of Buffered Solutions in Staining: Theory and Practice

The importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl₃, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.     

Induction of ketone body enzymes in glial cells

Ketone bodies serve a dual function in developing brain. They are important sources of energy for metabolism and serve as precursors for lipid synthesis. Astrocytes have two to three times higher activity than oligodendroglia for one of the enzymes involved in ketone body metabolism, 3-ketoacid-CoA transferase. Both glial cell types have similar levels of activity for beta-hydroxybutyrate dehydrogenase. Glucocorticoids and dibutytyl cAMP produce a significant stimulation of activity of both enzymes in astrocytes and oligodendroglia. However, the most striking induction in activity of the two enzymes is in the presence of hydrocortisone and sodium butyrate. There is a three- to eightfold stimulation with these effectors in both astrocytes and oligodendroglia. Thus, in brain the expression of ketone body enzyme activities is finely regulated by hormones and by agents that increase cAMP levels.     

The effect of methionine-sulphoximine on nerve and glia cells in vitro

Summary The effect of methionine-sulphoximine (MSI) on the behaviour of the nerve and glia elements from brain and cerebellum of rat and chick in tissue culture was studied. It was observed that a concentration of MSI^–3 M inhibits the growth of early brain expiants. Concentrations of 10^–4 and 10^–5 M did not show this effect. Time lapse cinematographic recordings evaluating continually the behaviour of living glial and neuronal elements in tissue culture showed that MSI in a concentration of 10^–3 caused, very shortly after administration, a conspicuous oedema of cellular elements. The highest degree of swelling occurred in the perikarya and processes of astrocytes. Cell bodies of the oligodendroglia swelled only in the beginning and later shrank again considerably. This shrinkage was accompanied by a retraction of the glial processes, their fragmentation, and destruction. The perikaryon of the oligodendroglia contracted discontinually. Similarly the volumetric increase of astrocytes was accompanied by rhythmic contractions. In neuronal as well as oligodendroglial perikarya movements of mitochondria and other cytoplasmic particles first accelerated, later slowed down. Synchronously with cytoplasmic swelling of neurons the formation of many vacuoles is observed. From a comparison of these changes, their time course, and the degree of damage in various cell types it may be concluded that MSI attacks in the first place glial elements, among them most intensely the oligodendroglia.     

Experimental Studies of Mechanisms Involved in Methods Demonstrating Axonal and Terminal Degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development or ¹¹⁰Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nuclei are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axon terminals in two separate staining procedures detailed in following papers.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Experimental studies of mechanisms involved in methods demonstrating axonal and terminal degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development of 110Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nucleic are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axons terminals in two separate staining procedures detailed in following papers.     

Staining Myelin in Brain with Gallein : A New Method

A procedure is described in which gallein, mordant violet 25, C.I. 45445, is used to demonstrate myelinated nerve fibers in animal brain. Specimens are fixed in 10% neutral buffered formalin and processed in a routine manner. Microsections are stained in an iron gallein solution with subsequent differentiation in 0.25% oxalic acid and 0.1% sodium carbonate solutions that avoid overdifferentiation. Methyl green is used to demonstrate other tissue elements. Myelin is stained deep violet, as are erythrocytes, with neuronal cell bodies and microglia shades of green. the staining procedure requires 30 minutes.     

Increase in Feulgen staining intensity by pre-treatment with different reagents.

In the experiments sections of rat tissues fixed in 10 per cent buffered neutral formalin for 3 hour and treated for 15 minutes with different chemical reagents such as pyridine, tributylamine, urea, tris sodium nitrite and sodium hydroxide were subjected to hydrolysis in 6 N HCl at 25 degrees C for 20 minutes and stained by the UV Feulgen technique. The results reveal a far more intense staining of the nuclei in tissues treated with any of the chemicals mentioned than in untreated controls. The possible role of these chemicals in enhancing the staining intensity is discussed.     

Staining myelin in brain with gallein: a new method.

A procedure is described in which gallein, mordant violet 25, C.I. 45445, is used to demonstrate myelinated nerve fibers in animal brain. Specimens are fixed in 10% neutral buffered formalin and processed in a routine manner. Microsections are stained in an iron gallein solution with subsequent differentiation in 0.25% oxalic acid and 0.1% sodium carbonate solutions that avoid overdifferentiation. Methyl green is used to demonstrate other tissue elements. Myelin is stained deep violet, as are erythrocytes, with neuronal cell bodies and microglia shades of green. The staining procedure requires 30 minutes.     

Regeneration of enzymatic activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and zinc acetate staining: the example of inositol 1,4,5-trisphosphate 5-phosphatase.

Regeneration of enzyme activity after sodium dodecyl sulfate-gel electrophoresis was investigated with a purified inositol 1,4,5-trisphosphate 5-phosphatase. In order to avoid silver or Coomassie blue staining, we have used zinc acetate. This staining procedure was sensitive, rapid, and reversible provided that zinc cations are chelated and activity is extracted after diffusion out of the gel. The method allows some gel lane staining and identification of the enzyme based on catalytic activity.     

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.     

UDP-Galactose:Ceramide Galactosyl Transferase of Isolated Oligodendroglia

Abstract: The activity of UDP-galactose:ceramide galactosyl transferase (CGalT) has been studied in isolated oligodendroglia from bovine brain white matter and myelinating rat brain. The specific activity and activity per mg DNA are 4- and 10-fold higher in rat oligodendroglia compared with neuronal perikarya from rat brain, and is higher in oligodendroglia from myelinating rat brain compared with bovine oligodendroglia. In membranes isolated from oligodendroglia, the specific activity decreased in the order endoplasmic reticulum > plasma membrane > myelin.     

Phase-partition fixation and staining of Drosophila eggs.

Aqueous solutions of alcohol-acetic acid-formalin or glutaraldehyde-acrolein were shaken with heptane and heptane phase used for fixation. Phase-partition fixation is akin to fixation with vapor. The organic solvent, immiscible with water, penetrates hydrophobic membranes and carries the fixative in contact with water phase of the tissue. Only the fixative enters the tissue, without changing the ionic and water-soluble substance concentrations in the tissue. The quality of this fixation for optical or electron microscopy was as good as that of any conventional fixation method. Staining with basic fuchsin after 2 N HCl hydrolysis gave brilliant staining of nuclei, more intense than that with Feulgen reagent, while cytoplasm remained nearly colorless. Fixing and staining procedures for Drosophila eggs are given.     

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.     

Properties of Bovine Oligodendroglia Isolated by a New Procedure Using Physiologic Conditions

A new class of procedures, previously shown to permit the isolation of pure oligodendroglia from whole rat cerebrum, has been applied with equal or greater success for the bulk isolation of this cell type from bovine white matter. Thus, the generality of this approach has been demonstrated. The bovine preparations have a purity of greater than 90% intact, phase-bright oligodendroglia and are obtained in a yield of 8 x 10(6) cells per gram of white matter. Within 1 day it is possible to obtain a preparation containing 60 mg of protein from a single cell type. These cells show a higher degree of ultrastructural preservation of all cytoplasmic constituents than previously obtained. The values for protein (33 pg/cell), DNA (5.4 pg/cell), and lipid (5-6 pg/cell) are very similar to those obtained with an earlier procedure. The cell lipids are rich in galactolipid, which comprises 20% of the total. The activity of the "myelin-specific" enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37), is 4.7 mumol/min/mg protein, similar to that obtained previously for isolated oligodendroglia and about 25-40% of that found in myelin. The activity of 5'-nucleotidase (EC 3.1.3.5) in the cells is about 10% of that in myelin or white matter.     

Selective staining of pericentromeric heterochromatin regions in chromosomes of spontaneously dividing cells with the use of the acridine orange fluorochrome

A novel phenomenon of unusual selective acridine orange (AO) staining ofpericentromeric heterochromatin regions (HRs) in chromosomal preparations from tissue with known spontaneous mitotic activity (chorionic villi, placenta, embryonic tissues, bone marrow, and testes), as well as embryonic stem cells, is described. Staining with 0.01% AO in a citric-phosphate (pH 5.5) or sodium phosphate (pH 7.0) buffer solution allows the HRs of human chromosomes (1q12, 9q12, 13p11.2, 14p11.2, 15p11.2, 16q11.2, 21p11.2, 22p11.2, and Yq12) and pericentromeric HRs of mouse chromosomes to be reliably detected by the red fluorescence of AO. This method of AO staining does not require any pretreatment. Explanations for metachromatic AO staining of polymorphic pericentromeric HRs in chromosomes of spontaneously dividing cells are suggested. A high reproducibility of the specific AO staining makes it possible to suggest its use as a reliable quick method for detection of polymorphic HRs of human chromosomes in cytogenetic prenatal diagnosis and oncohematology.