Phosphate ions in root-tip dividing cells: a combined trapping and squash method with implications for nuclear transcription.

We examined the pattern of inorganic orthophosphate (PPi) ion distribution in dividing cells of Zea mays root-tips. Unfixed and paraformaldehyde- or glutaraldehyde-vapor fixed tissues were immersed in lead acetate, glutaraldehyde, and cacodylate buffer to capture PPi as insoluble orthophosphate lead hydroxyapatite. Excess lead ions were removed with sodium citrate, then permeabilized in ammonia. Precipitates were stained with potassium sulfide, washed with distilled water and squashed in a drop of glycerin. The accumulation of PPi ions was cyclic in the cytoplasm during mitosis and they surrounded all chromosomes during metaphase and anaphase. Partition between dividing cells started with a high concentration of PPi ions at sites where plasma membrane and cell walls formed. Small daughter cells and those in G1 phase had PPi concentrated in the nucleolus, with lower levels elsewhere in the nucleus. Later in the cell cycle, there were greater amounts of PPi ions associated with condensed chromatin in larger nuclei. In Xenopus laevis oocytes, PPi was concentrated in the nucleus, mainly in the active central core of multiple nucleoli. These results and others indicate that compartmentalization of PPi occurs in the intact cell and correlates with the rate of transcription in distinct functional domains within the nucleus.

Differential staining of two subpopulations of Purkinje neurons in rat cerebellum with acid dyes.

We present a new method that stains differently two subpopulations of Purkinje cells in the adult rat. Deparaffinized sections of cerebella, fixed by perfusion with buffered glutaraldehyde or Bouin's fluid were stained with 0.5% light green in 50% ethanol (10-30 min). The excess dye was removed with saturated aqueous picric acid (10-30 min). At this point some Purkinje cells appeared as lightly stained neurons, while others were strongly stained. Slides were immersed in 0.5% aqueous acid fuchsin for approximately 1 min until the lightly stained neurons acquired a red color. Following immersion in 1% phosphotungstic acid, slides were rapidly dehydrated in ethanol, passed to xylene and mounted in Canada balsam. Two subpopulations of Purkinje cells differing in their protein content in somata and proximal dendrites stained differentially by this method. They occurred in all coronal and sagittal sections and in patches or stripes. Their relative proportion varied from lobule to lobule. A second staining method used potassium permanganate as the sole staining reagent. The staining reagent can be used on sections previously stained with the acid dyes. Purkinje cells appeared as subsets of brownish to deep brown stained neurons, the latter ones corresponding to green stained cells in the dichromic method. The results obtained indicated that the subpopulations reflect real differences among individual neurons and are not artifacts. The technique holds promise for identifying and localizing sub-sets of Purkinje cells differing in their protein content under normal and experimental conditions and for their further characterization by combined staining and histochemical procedures.

Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method.

The immunofluorescence technique is one of the most useful methods for localizing antigens in several tissues, including the central nervous system. For immunohistochemical procedures, especially immunofluorescence methods, formaldehyde is commonly used as a fixative agent. But for some protocols, mainly in neurobiology, glutaraldehyde is necessary to recognize a number of small molecules (haptens) whose antisera have been raised using glutaraldehyde as the cross-linking agent. This is a severe limitation because glutaraldehyde gives rise to a strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. In this paper we present a new method that allows the use of immunofluorescence techniques on glutaraldehyde-fixed tissues. The new method consists of a treatment of tissue sections with the Schiffs reagent (leucobasic fuchsin) followed by a reduction of the Schiff-dye with sodium borohydride. This reduced dye produces a quenching of glutaraldehyde-induced fluorescence on the tissue. The goal of the new method is to make possible the use of a great number of available glutaraldehyde-raised antisera for immunofluorescence techniques, a useful tool in both basic and clinical research.

Selective axonal argentaffin staining in rat central nervous system after protein mercuration.

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.

Presence of a centromeric filament during meiosis.

Spermatocytes at meiotic metaphase I and anaphase I have a characteristic centromeric filament in a variety of vertebrate organisms. This centromeric filament was first demonstrated on mouse spermatocytes and its presence is now extended to spermatocytes from the human, rat, golden hamster, bull, and chicken. The visualization of this filament was possible through the use of a novel silver-staining technique, which allows a high contrast between the filament and the centromeric chromatin. In the species cited, the centromeric filament shares an intense staining, a short (0.2-0.6 micron) length, a curved and branched shape, and location inside the centromeric chromatin of seemingly every homologue of the complement. The similarity of staining reactivity and the observation of transitional structures during first meiotic prophase strongly suggest that the centromeric filament is a remnant of a lateral element of the synaptonemal complex, which stays specifically at both centromeric regions of each bivalent. This filament is not found at the second meiotic division or at the centromeres of mitotic chromosomes. It is assumed that this centromeric filament joins the two sister chromatids of each homologue at the centromere and thus ensures the proper coorientation of sister kinetochores at metaphase I. Further testable assumptions on the functions of this filament are presented.

A specific ultrastructural method to reveal DNA: the NAMA-Ur.

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

Selective extraction, solubilization, and reversed-phase high-performance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures.

The number of solvents capable of dissolving myelin and proteolipid protein (PLP) and of being used as a mobile phase for the separation of myelin proteins by reversed-phase high-performance liquid chromatography (RP-HPLC) is very limited. In a thorough study, we found that aqueous tetrahydrofuran (THF) fulfilled such a requirement. The maximal amount of protein extracted corresponded to a THF/water ratio of 4:1 v/v and a polarity index of 5.16. This mixture dissolved a purified PLP preparation completely, 60% of proteins from fresh myelin, and 20% of white matter total proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of those extracts, followed by densitometric analysis, showed that the amount and type of proteins dissolved depended on the polarity (i.e., the content of water) of the solvent mixture used. This selective effect was greater for basic protein in myelin preparations. Crude extracts highly enriched in basic protein can be prepared. In addition, the solvent system THF/water proved to be very useful as a mobile phase in RP-HPLC for separating myelin proteins. Using a C3 column and a linear gradient from 30% to 100% THF in water, both containing 0.1% trifluoroacetic acid (TFA), we separated completely the three main protein fractions of central nervous system (CNS) myelin in a short period of time. The high solubility power of THF/water mixtures prolonged greatly the life of the column.

Alkaline hydrolysis/methylation-acetylation: a new technique for ultrastructural DNA cytochemistry.

A new technique for the visualization of DNA-containing structures in electron microscopy is described. Samples of glutaraldehyde-fixed bone marrow from rats were subjected to alkaline hydrolysis to remove RNA and the phosphate of phosphoproteins, followed by a combined blockage of protein carboxyl and amino groups through methylation-acetylation. After uranyl acetate staining of epoxy-embedded ultrathin sections, chromatin from all cell types showed a highly selective and intense electron opacity. Staining methods for DNA were also positive in semithin sections. This simple procedure could be very useful in ultrastructural cytochemistry of DNA and chromatin.

An 'axis-like' material in the centromeric region of metaphase-I chromosomes from mouse spermatocytes.

This study reports the persistence of axis-like structures in the centromeric region of both homologues during the metaphase-I and anaphase-I stages of meiotic division of mouse spermatocytes. A novel type of silver 'argentaffin' technique (NH4-Ag) is employed. This technique includes the treatment of glutaraldehyde-fixed tissues with dilute ammonium hydroxide followed by a reduction of aldehyde groups with sodium borohydride. Staining is accomplished with ammoniacal silver nitrate in darkness followed by sulfite washing. The lateral elements of synaptonemal complexes and the single chromosomal axes of diplotene spermatocytes show a prominent reactivity with this technique. The pattern of very small grains over condensed chromatin is uniform and gives only a light opacity to the electron beam. The presence of an axis-like structure is seen in every centromeric end of meiotic chromosomes at metaphase I and anaphase I. The chromatin (heterochromatin) that surrounds the centromeric filament and some material distributed in irregular linear arrays along some of the homologues also showed a higher electron opacity than the bulk of deoxyribonucleoprotein. While the former is related to C+ heterochromatin, the latter could represent dispersed material of diplotene axes. It is suggested that the disposal of axial material is differentially delayed at the centromeric regions. The present evidence supports the hypothesis that axial fragments or lateral-element segments persisting at these regions contribute to the cohesiveness of centromeres of sister chromatids during normal disjunction.

A silver-reducing component in rat striated muscle. I. Selective localization at the level of the terminal cistern/transverse tubule system. Light and electron microscope studies with a new histochemical procedure.

This study reports the presence of a silver-reducing constituent in rat striated muscle fiber located selectively at the level of the terminal cistern/transverse tubule system. It is related to the T tubule network at or near sites that participate in junctions with terminal cisternae, i.e., at both sides of the T tubule in skeletal muscle (triad) and, predominantly, at one side in the ventricle (dyad). Little reactivity is present in the auricle due to the scarcity of those membrane systems. The longitudinal sarcoplasmic reticulum, the sarcolemma, mitochondria and myofibrils are not outlined by the reaction product. Extraction of low molecular weight substances, nucleic acids and lipids did not suppress the chemical reaction. A new argentaffin (Hg--Ag) technique is described. Ethanol or aldehyde fixed muscles were passed to water, postfixed 6-24 h with mercuric acetate (5% w/v in 1% acetic acid), washed with 1% acetic acid and distilled water, stained 12-24 h at 43 degrees C with ammoniacal silver nitrate (60% w/v) and washed in 10% sodium sulfite (three changes) and water. All steps were carried out in darkness. Postfixation with mercuric acetate proved to be essential for immobilizing the argentaffin component without interfering with its strong argentaffinity. The procedure also provides a simple method for tracing the pathway of transversally oriented membrane systems in skeletal and cardiac muscle cells.

A silver-reducing component in rat striated muscle. II. Isolated sarcoplasmic reticulum vesicles.

In previous work on rat striated muscle cells a silver-reducing component was found selectively localized at the terminal cistern/transverse tubule system (Tandler and Pellegrino de Iraldi 1989). To further investigate that problem we performed the Hg-Ag argentaffin reaction on a sarcoplasmic reticulum fraction from rat skeletal muscle. Circular profiles corresponding to vesicular structures were found outlined by silver grains. The number of silver "stained" vesicles were less than the total number vesicles stained by conventional procedures. The correlation between argentaffinities in the intact muscle fiber and their subcellular organelles indicated that the Hg-Ag reactive vesicles must be those derived from the terminal cisternae of the sarcoplasmic reticulum. The silver-reducing constituent aggregates in the presence of 1 mM CaCl2 or 0.5 M K cacodylate. The state of aggregation induced by Ca2+ was not affected by incubation with 0.5% Triton X-100 or by 2 mM EDTA, thus suggesting a localization at or near the membrane of the terminal cistern vesicle facing the junctional gap. In Laemmli SDS-acrylamide gels the Hg-Ag reaction stained all proteins in a manner similar to Coomasie blue. It is suggested that the selective histochemical staining is the result of differential reactivities due to steric requirements of the chemical reaction.

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Existence of cyclic secondary amino groups in the immunogen.

The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro-beta-carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.

Fine structure and cytochemistry of the mitotic plaques of Trypanosoma cruzi and Crithidia fasciculata.

The mitotic plaques are double, electron-dense structures which are located at the equator of the nucleus during the equatorial (metaphase) stage of mitosis in Trypanosoma cruzi, Crithidia fasciculata and other trypanosomatids. Each part of the equatorial plaques separates from the other and becomes an hemiplaque at the beginning of nuclear elongation. Variations of size of the plaques in different species of Trypanosomatidae are restricted to a limited range (less than 30% of the average thickness). At least two different components are found in the plaques with cytochemical methods: a) a basic protein with a high affinity for ethanolic-phosphotungstic acid, which is located in a narrow band towards the cleavage plane in each hemiplaque; and b) an osmiophilic component (possibly a protein) with a low affinity for uranyl acetate and which is located throughout the body of the plaque. The affinity for uranyl acetate can be abolished by methylation and acetylation, but remains after extraction with cold perchloric acid. No cytochemical evidence for the presence of DNA in the plaques is found. However, electron microscopy and cytochemical observations show that the PTA-affine band of the plaques is associated at its sides with chromatin fibers. Thus plaques, as the outer layer of kinetochores in higher eukaryots, have a component with high affinity for phosphotungstic acid, strengthening the hypothesis that these structures are phylogenetically related.

Methanol-acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues.

A mixture of absolute methanol and acetic anhydride (MA) (5:1, v/v; 24 h at 25 degrees C) which is known to methylate and acetylate--respectively--free carboxyl and amino groups in proteins, was tested for its effectiveness as a blocking agent in glutaraldehyde-fixed mouse testis and skeletal muscle. In young spermatids the staining of the acrosome with either uranyl acetate (UA) or ethanolic phosphotungstic acid (PTA) was completely prevented by prior treatment with MA. Esterification of carboxyl groups with 0.1 N HCl in methanol (24 h at 30 degrees C) eliminated the UA staining without affecting that due to PTA. It is concluded that--COOH groups are responsible for UA binding and that acetylation (of amino groups) prevented PTA binding. MA also abolishes the strong affinity of PTA to the lateral elements of the synaptonemal complex in meiotic chromosomes, the axoneme and fibrous sheath of the spermatid tail and the Z band in skeletal muscle. Reactivity was diminished in nucleoli and remained unaffected in chromatin, the outer coarse fibers of the flagellum and collagen fibrils. Different functional groups may participate in PTA staining. The ultrastructure was well preserved in all cases.

Dithiocarbamylation in histochemistry: carbon disulfide as a reagent for the visualization of primary amino groups with the light and electron microscope.

Tissue amino groups undergo dithiocarbamylation on treatment (12 hr, 22-25 degrees C) with a carbon disulfide:triethylamine:tetrahydrofuran (1:1:0.5, v/v) mixture. After washing with tetrahydrofuran:triethylamine (4:1, v/v) followed by tetrahydrofuran:pyridine (4:1,v/v) and ice-cold distilled water tissues were immersed into ice-cold 10% (w/v) lead acetate to yield yellowish lead dithiocarbamates. At 22-25 degrees C the derivatives obtained from primary amines decompose to lead sulfide. After washing with 5% lead acetate in 10% acetic acid followed by three changes of distilled water tissues are dehydrated and embedded in paraffin or Maraglas as usual. In proteins the reaction is given by the epsilon-amino group of lysine and alpha-terminal amino groups, while nucleic acids do not react. Results are shown in tetrahydrofuran-delipidized ethanol or formalin-fixed 1) plant root tips where brown-colored and electron-dense lead sulfide deposits are localized in condensed chromatin and chromosomes attributable to lysine-rich histones and 2) rat skeletal muscle where an interfibrillar component is visualized in a repeating pattern, possibly related to terminal regions of the sarcoplasmic reticulum.

Inorganic cations in the cell nucleus. Selective accumulation during meiotic prophase in mouse testis.

Earlier reports indicated the presence of significant amounts of inorganic salts in the nucleus. In the present study the possibility that this might be related to the transcription process was tested on seminiferous epithelium of the adult mouse, using potassium pyroantimonate as a fixative. The results indicated that a correlation exists between the inorganic cations comprising the pyroantimonate-precipitable fraction and the RNA synthetic activity. During meiotic prophase an accumulation of cation-antimonate precipitates occurs dispersed through the middle pachytene nuclei, the stage in which RNA synthesis reaches a maximum. At other stages (zygotene to diplotene), where RNA synthesis falls to a low level, that pattern is not seen; cation-antimonate deposits are restricted to a few masses in areas apparently free of chromatin. The condensed sex chromosomes, the heterochromatin of the "basal knobs," the axial elements, and the synaptonemal complexes are devoid of antimonate deposits during the meiotic prophase. The Sertoli cells, active in RNA synthesis in both nucleoplasm and nucleolus, show cation-antimonate deposits at these sites. In the nucleoplasm some "patches" of precipitates appear coincident with clusters of interchromatin granules; in the nucleolus the inorganic cations are mainly located in the fibrillar and/or amorphous areas, whereas relatively few are shown by the granular component. The condensed chromatin bodies associated with the nucleolus were always free of antimonate precipitates. It is suggested that the observed sites of inorganic cation accumulation within the nucleus may at least partially indicate the presence of RNA polymerases, the activity of which is dependent on divalent cations.

Inorganic cations in rat kidney. Localization with potassium pyroantimonate--perfusion fixation.

For localization of pyroantimonate-precipitable cations, rat kidney was fixed by perfusion with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative). A remarkably good preservation of the tissue and cell morphology was obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. All proximal and distal tubules and glomeruli were delimited by massive electron-opaque precipitates localized in the basement membrane and, to a lesser extent, in adjacent connective tissue. In the intraglomerular capillaries the antimonate precipitate was encountered in the basement membranes and also between the foot processes. In addition to a more or less uniform distribution in the cytoplasm and between the microvilli of the brush border, antimonate precipitates were found in all cell nuclei, mainly between the masses of condensed chromatin. The mitochondria usually contained a few large antimonate deposits which probably correspond to the so-called "dense granules" observed after conventional fixations.