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PubMed1980

An ultracytochemical study of nucleolar organization in meristematic plant cells (Allium porrum).

The interphase nucleolus in Allium porrum, as in many of the plant species studied so far, is highly heterogeneous in ultrastructure owing to the presence of coarse, contorted, thread-like structures, or nucleolonemata. Each nucleolonema appears to be sharply twisted and to give rise to a skein within the nucleolar mass. In order to characterize further these nucleolar components, a variety of cytochemical techniques were exploited. For that purpose, specimens were mostly fixed in 4% formaldehyde and stained in the block according to procedures known to reveal the presence of nucleic acids or proteins. Certain specimens were also digested with deoxyribonuclease, ribonuclease or proteinase K before staining. By staining with phosphotungstic acid or bismuth oxynitrate, the presence of a high concentration of proteins can be demonstrated within thin (0.15 micrometer), filamentous structures which are believed to correspond to the outer region of the nucleolonema. Such convoluted formations disappear upon sufficiently long extraction with proteinase K. Using Bernhard's regressive staining technique for chromatin, the distribution of this substance throughout the nucleolar mass was found to match closely that of the nucleolonemata as revealed by several other procedures. As a last test for investigating the cytochemical make-up of the nucleolus, blocks of tissues were stained with 3,3'-diaminobenzidine, a substance known to react specifically with nucleic acids. When such specimens are digested with ribonuclease for 1 h, there persist within the nucleolus, fibrillogranular zones the localization of which is highly reminiscent of that of the nucleolonemata. Combination of ribonuclease hydrolysis with subsequent treatment with proteinase K (30 min) induces the extraction of a large proportion of the nucleolar material, the persisting loose and rather evenly distributed fibrils exhibiting a diamter of 3-5 nm. The possibility is considered that these units may correspond to chromatin fibrils although they have most likely been displaced from their original localization during the extraction procedures. Our cytochemical data suggest that, in Allium porrum, the nucleolonema is approximately 0.3 micrometer in diameter and may consist of a central axis from which chromatin loops project radially. A possible interpretation for the presence of protein-rich, 0.1 micrometer-thick, annular structures throughout the nucleolonemal skein is that the newly synthesized RNP products are accumulated transiently at the extremities of these loops before migrating to the immediately adjacent granular nucleolar zones.

Cell Nucleolus↗

PubMed1997

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.

Animals↗

PubMed1976

[Acid mucopolysaccharide detection on ultra-thin sections by alcian blue in dental tissues embedded in Epon].

Ultra-thin sections of dental tissues, fixed in glutaraldehyde and osmium tetroxide, embedded without previous demineralization in Epon, were contrasted the grids with an alcian blue solution after previous oxidation periodic acid or hydrogen peroxide. Correlations with histochemical results obtained with the optical microscope, other results obtained with ruthenium red and phosphotungstic acid and controls made after enzymatic digestion with chondroitinase AC suggest that this technique is specific for the detection of acid mucopolysaccharides in those calcified tissues studied.

Animals↗

PubMed1978

The structure of the tips of mammalian respiratory cilia.

The ciliary crown and the relationship of the ciliary crown to the underlying axoneme were studied by electron microscopy in cilia from hamster and rat trachea and bronchioles, and rabbit trachea. The ciliary crown is a cluster of 4 to 6 fibrils 35 nm long protruding beyond the plasma membrane at the tips of the cilia. The fibrils are well preserved after tannic acid-glutaraldehyde-osmium tetroxide fixation and have high contrast with a periodic density of 4.5 nm. They stain relatively weakly with phosphotungstic acid. The surface of the fibrils stains with ruthenium red. The microtubules of the axonemem end in a plate of electron dense amorphous material. A five layered disc occupies the space between the membrane and the amorphous plate at the tip of the axoneme. The plasma membrane can be dissolved with the detergent triton X-100 without loss of the ciliary crown. This indicates that the ciliary crown is composed of transmembranous filaments which are bound to the disc at the tip of the axoneme.

Animals↗

PubMed1995

[Ascorbic acid concentrations in ruminal fluid and in blood of cattle and sheep].

Plasma and ruminal fluid ascorbic acid levels were determined in Holstein calves aged four months and in ruminal fluid of merino rams aged 2,5 years under in vitro conditions. Ascorbic acid values decreased in plasma until 6th month. The highest Ascorbic acid value in ruminal fluid was found in 5th month. Under in vitro conditions shown ascorbic acid concentrations in ruminal fluid with time upward tendency. For analysis of ascorbic acid concentrations the acid phosphotungstate method were used for each samples.

Aging↗

PubMed1983

Fibrin linkage: a precursor for new attachment.

Previous studies indicated that connective tissue attachment to the root surface appeared to be dependent upon a chronologic healing sequence related to fibrin and collagen interactions. It was the purpose of the present study to try and substantiate this hypothesis by using histologic techniques designed to differentiate between fibrin and collagen during healing at the root surface interface. In four squirrel monkeys, 24 normal teeth were extracted and reimplanted after either (i) surgically denuding the coronal root surface of connective tissue fibers and cementum by root planing or, (ii) surgical denudation followed by topical application of citric acid (pH = 1; 3 minutes). Three specimens were available for histological analysis 1, 3, 7 and 21 days after reimplantation. Mallory's phosphotungstic acid hematoxylin staining technique was used to differentiate between fibrin and collagen. Epithelium migrated rapidly along the denuded, non-acid-treated, root surfaces, had reached the alveolar crest at 3 days, and was within the ligament space to the level of root denudation at 21 days. Epithelium did not migrate apically along denuded root surfaces treated with citric acid. At 1 and 3 days, inflammatory cells were enmeshed in a fibrin network which appeared to be attached to the root surface by arcade-like structures. At 7 and 21 days, the region had repopulated with connective tissue cells, and collagen fibers had replaced the fibrin. It was concluded that collagen fiber attachment to the root surface was preceded by fibrin linkage, and that the linkage process occurred as an initial event in the wound healing response.

Animals↗

PubMed1989

Detection of cationic and non-cationic markers in the rat glomerulus by electron probe analysis.

Acidic glycans (glomerular polyanion substances) in the rat kidney were visualized ultrastructurally by three cationic markers: colloidal iron, ruthenium red, and polyethylenimine-phosphotungstic acid (PEI-PTA). Heavy metal atoms (Fe, Ru and W) were detected in ultrathin sections by energy-dispersive electron probe microanalysis (EPMA). Characteristic peaks of the locally bound elements were obtained in spectra derived from the dense structures seen by transmission electron microscopy (TEM)--i.e. the glycocalyx of podocytes and/or the polyanion sites in the lamina rara externa of the glomerular basement membrane. Weaker signals were emitted by some extraglomerular structures. This finding may reflect a low concentration of glycans in structures lacking apparent density by TEM, and/or incomplete specificity of the markers, partial dislocation of reactive substances or the presence of an endogenous element (Fe). Experimental argyrosis was elicited by the peroral administration of silver nitrate. Dense Ag precipitates were seen chiefly in the lamina densa and characteristic peaks of silver were displayed in this site by EPMA, and was best demonstrated in non-contrasted sections. A single i.v. injection of Ag proteinate failed to produce glomerular pigmentation. The only dense granular product in tubular cells yielded characteristic peaks of Fe (endogenous siderosomes) but EPMA excluded detectable amounts of silver.

Animals↗

PubMed1988

[The use of ribonucleic acid as a marker for measuring microbial protein yield in the rumen. 1. Chemical determination of ribonucleic acid].

The determination of ribonucleic acid (RNA) in the duodenal digesta and the rumen microbes was carried out colorimetrically with orcinol after extraction with hot NaCl solution, precipitation with phosphotungstic acid and alkaline hydrolysis. 96 +/- 1% of the RNA supplemented to the digesta content was recovered (y +/- s, n = 5). The methodic error (repetition accuracy) was 1.1%. The relation of RNA-N:N (mg/g) in the bacteria mass amounted to 93.8 +/- 3.99 (y +/- s(-y), n = 48) and to 16.1 +/- 0.8 (y +/- s(-y), n = 51) mg RNA/g DM in the duodenal content.

Animals↗

PubMed1993

Use of tannic acid and silver enhancer to improve staining for electron microscopy and immunogold labeling.

Staining by uranyl acetate and lead citrate (UA-LC) of immunolabeled sections of unfixed glomerular basement membrane (GBM) digests embedded in low-acid glycol methacrylate (LA-GMA) is poor. The following were investigated for their ability to enhance contrast when applied to sections before UA-LC: potassium permanganate, phosphotungstic acid, gold chloride, osmium tetroxide, glutaraldehyde-osmium tetroxide, colloidal gold-silver enhancer, tannic acid, and glutaraldehyde-tannic acid. Silver enhancer (2 min incubation, no sodium thiosulfate step) gave dense GBM staining but not with immunogold labeling. Silver enhancer is recommended as a simple alternative to routine silver stains but not for contrast enhancement with immunogold labeling. Tannic acid (1% for 1 min or 0.005% for 10 sec) and glutaraldehyde (2.5% for 5 min) followed by tannic acid enhanced contrast and, when applied after immunolabeling, did not appear to affect probe levels. Tannic acid also enhanced the staining of fixed tissue in LR Gold acrylic resin and LA-GMA, but not of glutaraldehyde-osmium-fixed tissue in epoxy resin, even after sodium metaperiodate treatment. Treatment of sections with tannic acid is recommended for contrast enhancement in immunogold studies when osmium and tannic acid post-fixation must be avoided and the tissue is embedded in methacrylate or acrylic resin.

Basement Membrane↗

PubMed1980

Lysergic acid diethylamide: morphological study of its effect on synapses.

A morphometric analysis of the effect of LSD on synapses was performed in the habenulae and the interpeduncular nucleus of the frog. The OsO4-fixed nervous tissue was treated with ethanol phosphotungstic acid (EPTA). Intersection of lattice lines with synapses were counted as a proportion of the total length of lines. LSD-treated frogs had a higher total area of synaptic contact than control frogs. Exocytosis profiles were observed only in LSD-treated frogs Other qualitative changes in the ultrastructural characteristic of synapses were appreciable after LSD administration.

Animals↗

PubMed2006

[Effects of different durations of thromboembolism on blood gases, hemodynamics, pulmonary arteriography and thrombo-pathology in a canine model with selective embolization of pulmonary lobar arteries].

OBJECTIVE: To investigate the effects of different durations of thromboembolism on blood gases, hemodynamic parameters, pulmonary arteriography and thrombo-pathology in an animal model mimicking chronic pulmonary thromboembolism (PTE). METHODS: Sixteen dogs were embolized with five thrombi developed by autologous blood into the left lower pulmonary artery (n = 15) and the right lower pulmonary artery (n = 1, used to confirm the available method of selective embolization). The 15 dogs were divided into three groups: sham group (n = 5), one-week group (n = 5) and two-week group (n = 5) according to the different durations of embolization. Swan-Ganz catheter was used to guide a plastic duct, through which the thrombi were injected selectively into the left or right lower pulmonary artery by X-ray fluoroscopy. Local pulmonary arteriography of lower pulmonary arteries was taken. Blood pressure (BP), and blood gases were measured. Central venous pressure (CVP), mean pulmonary arterial pressure (MPAP), pulmonary arteriole wedge pressure (PAWP), and cardiac output (CO) were recorded, and pulmonary vascular resistance (PVR) was calculated. Each dog underwent muscular injection with tranexamic acid for one or two weeks to prevent thrombolysis. The lower lung lobe was dissected to confirm the thromboembolism after one or two weeks. The lung sections were stained with phosphotungstic acid hematoxylin (PTAH) to observe thromboemboli with optical microscopy. RESULTS: In the PTE group, PaO(2)/FiO(2), MPAP and PVR changed significantly as compared to baseline values (P < 0.05) after one hour of embolization, with MPAP increasing from (15 +/- 3) mm Hg to (21 +/- 4) mm Hg, PVR increasing from (178 +/- 114) mm Hg.s/L to (404 +/- 260) mm Hg.s/L, and PaO(2)/FiO(2) decreasing from (508 +/- 58) mm Hg to (395 +/- 100) mm Hg; these parameters returned to the baseline values one or two weeks later. After embolization, pulmonary arteriography demonstrated lower lobar artery cut-off perfusion defects. One week later, pulmonary arteriography demonstrated irregularities and stiffness of the arterial wall, enlarged proximal part of lower pulmonary artery and cut-off perfusion defects. Poor filling at embolus site was evident after embolization for two weeks. In the 1-week PTE group, organized tissue covered with the blue-purple fibrin nest was observed in the thrombus with PTAH stain. In the two-week group, the well organized thrombi were partially recanalized and surrounded and invaded by hyperplastic tissues from pulmonary artery wall. CONCLUSIONS: A canine model mimicking chronic PTE can be established by the use of fibrinolytic inhibitor tranexamic acid. Different manifestations on pulmonary arteriography and varied degree of organization of thrombi are evident at different times after embolization.

Angiography↗

PubMed1975

The chemical nature of human atrial specific granules.

The nature of human auricular specific granules was assessed by a variety of cytochemical and histochemical methods. The specific granules were found to be argentaphobic when ultrathin sections of Araldite-embedded auricular appendages were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of these granules was moderately positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded auricles were stained with phosphotungstic acid (PTA) at a low pH. A similar reaction was shown by the cell coat, residual bodies (C-granules), lysosomes, Z-discs as well as by a very small portion of the Golgi complex. Analogous results were obtained in semithin sections of GMA-embedded auricles stained by the periodic acid-Schiff (PAS) technique. Incubation of ultrathin sections (fixed in glutaraldehyde and embedded in GMA) with proteolytic enzymes (pronase, pepsin, trypsin, or alpha-chymotrypsin) elicited selective digestion of atrial specific granules and Z-bands and, to a much lesser degree, of the cell coat. It is concluded that human auricular specific granules, as in rat atrial cardiocytes, are composed mostly of proteins. In addition, these granules may contain complex carbohydrates.

Chemical Phenomena↗

PubMed1989

[Modification of the determination of lipid peroxidation products in a reaction with thiobarbituric acid].

The aim of the study was to choose the optimal conditions for spectrophotometry of the blood serum lipid peroxidation (LPO) products by the thiobarbituric acid (TBA) test. LPO products have been measured in trichloroacetic acid filtrate and in lipoprotein sediment by spectrophotometry and fluorometry. The optimal conditions for spectrophotometry of TBA-active LPO products are as follows: the proteins are sedimented with 20% solution of phosphotungstic acid, stabilizing the pH values in 1.3-1.5 range; the incubation temperature is 99-100 degrees C; the optic density of the aqueous extract is measured spectrophotometrically at wavelengths 535 and 580 nm. The suggested method permits spectrophotometry of LPO products without additional extraction with butanol, and its results are compatible with those of fluorometry. The coefficient of convergence variations is 3.42%, the repeatability factor 3.31%.

Humans↗

PubMed1980

Ultrastructural cytochemistry of atrial and ventricular cardiocytes of the bullfrog (Rana catesbeiana). Relationship of specific granules with reninlike activity of the myocardium.

The ultrastructural cytochemical reactivity, renin activity, and cathepsin D activity of atria and ventricle of the bullfrog have been assessed. The specific granules (A, B, and D) were found to be argentaphobic when ultrathin sections of Araldite-embedded atria and ventricle were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of the specific granules was moderated positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate embedded atria and ventricle were stained by phosphotungstic acid at a low pH. A similar reaction was shown by the cell coat, intercalated discs, residual bodies (C granules), and Z discs as well as by a very small portion of the Golgi complex. Incubation of ultrathin sections of atria and ventricule fixed only in glutaraldehyde and embedded in glycol methacrylate with either pronase or trypsin resulted in selective digestion of specific granules and Z discs and, to a much lesser degree, of the cell coat. As cathepsin D activity and renin activity were present in both atria and ventricle, the generation of angiotensin I by these cardiocytes might have been due to either enzyme. Nevertheless, because of the glycoprotein nature of specific granules and of the endocrinelike ultrastructure of atrial and ventricular cardiocytes in the frog, the present results raise the possibility that specific granules may contain renin.

Animals↗

PubMed1975

Chemical nature of atrial specific granules.

The specific granules are argentafugic when ultrathin sections of Araldite-embedded atria are stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of the atrial specific granules is moderately positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate- (GMA-) embedded atria are stained with phosphotungstic acid at a low pH. A similar reaction is shown by the cell coat, intercalated discs, residual bodies (C-granules), and Z-discs, as well as by a very small portion of the Golgi complex. Analogous results are obtained with semithin sections of GMA- embedded atria stained according to the periodic acid-Schiff (PAS) technique. In ultrathin sections of GMA-embedded atria stained with dialyzed colloidal iron (DI), the cell coat of the cardiocytes is positive, unlike all the other cytoplasmic organelles. When ultrathin sections of GMA-embedded atria are incubated with proteolytic enzymes (pronase, pepsin, or trypsin), atrial specific granules and Z-bands and, to a much lesser degree, cell coat and sarcolemma are selectively digested. Proteins are also distinctly demonstrated in the paranuclear specific granules by a variety of histochemical techniques. These results indicate that atrial specific granules are rich in proteins and possess a weak complement of complex carbohydrates.

Animals↗

PubMed1977

Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries, and arterioles of the ischemic (endocrine) kidney. An ultrastructural-cytochemical and autoradiographic study.

Partial ligation of the aorta between the renal arteries induces marked atrophy of the cortical tubules of the left (endocrine) kidney with a remarkable increase in the number and granularity of hypersecretory juxtaglomerular cells (JGC), which are found not only at the glomerular pole of arterioles but also in the walls of arteries and arterioles far removed from the glomerulus. Typical vascular smooth muscle cells (SMC), in which secretory granules appear, show a concomitant development of their Golgi complex and rough endoplasmic reticulum, with a gradual decrease in the number of their filaments. Microtubules also appear in the Golgi area. Thiery's periodic acid-thiocarbohydrazide-silver proteinate technique demonstrates that in these "intermediate" cells, as in mature JGC, the amount of glycogen is greater than in SMC. The newly-developed secretory granules of intermediate cells are stained by phosphotungstic acid at a low pH, as are the mature granules of JGC, an indication that both types contain glycoproteins. Light and electron microscopic autoradiography reveal that both JGC and "intermediate" cells of the vascular wall do not incorporate radioactive thymidine (injected during the 10-day observation period). Thus, they develop by metaplasia of preexistent SMC. In control kidneys, radioactive thymidine is practically never incorporated into the nuclei of SMC but is found in a few glomerular and tubular cells of all zones except the papilla.The endocrine kidney shows virtually no reactive nuclei in vascular SMC, glomeruli, or tubular cells of the outer cortex. Thymidine is incorporated into practically all nuclei of the straight portion of proximal tubules and into about half the nuclei of all medullary tubular cells including the papilla.

Animals↗

PubMed1976

Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation".

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.

Animals↗

PubMed1960

The fine structure of Meissner's touch corpuscles of human fingers.

Thin slices of the finger pads of six individuals were fixed in buffered 1 per cent osmic acid, embedded in deaerated, nitrogenated methacrylate, and cut into thin sections for electron microscopic study. Before embedding, the slices were trimmed so as to include several digital tactile corpuscles. Some thin sections were stained in 10 per cent aqueous phosphotungstic acid solution. The principal part of Meissner's corpuscle is made up of flattened laminar cells stretching across the corpuscle in irregular layers. The perinuclear cytoplasm of these cells contains numerous small mitochondria, a sparse granular endoplasmic reticulum, and a large number of small vesicles. Nerve fibers enter the side or base of the corpuscle, lose their myelin sheaths, and follow a meandering course between the laminar cell plates. The nerve endings enter into a close appositional relationship with the flattened portions of the laminar cells. In some areas the apposed axolemma and cell membranes are slightly thickened with small vesicles located along the cell membrane or on both surfaces. These regions are interpreted as synapses. The most prominent feature of the nerve endings is an extraordinary accumulation of small mitochondria which vary in size and internal density. The nerve endings also contain vacuoles, groups of dense concentric membranes, and small dense vesicles of irregular distribution. The laminar cells are separated from one another by a dense intercellular substance of uniform thickness which also envelops the entire corpuscle. This material contains randomly oriented collagen fibers and fine fibrils bound together by a dense material at nodal points recurring at regular intervals of approximately 120 mmicro. These findings are discussed in relation to the problems of the function of Meissner's corpuscle, neural material loss and replacement, and the presence of synapses.

Animals↗