Method for additional electron-dense contrast enhancement of nucleic acids in nucleus and cytoplasm accompanying histochemical detection of sodium cations in cell and tissue ultrastructures


G01N1/06 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biology, namely to a method for additional electron-dense contrast enhancement of acid groups of biomolecules accompanying the histochemical detection of sodium cations in pulmonary and tracheal cell and tissue ultrastructures. Substance of the method consists in fixing tissue slices, washing the surface in bidistilled water, placing into a diluted agent containing 4% osmium tetraoxide 1 ml and 2% potassium hexahydroantimonate 8 ml. The tissue slices are stained for 4 hours while stirring strongly and washed in bidistilled water. The tissue slices are further prepared by slicing thinner and dehydrated; the semi-thin and ultrathin slices are produced and studied by a transmission electron microscope, which is followed by computer processing to detect diffuse selective staining of the acid cell ultrastructures and intracellular substance.

EFFECT: using the declared method enables the additional electron-dense contrast enhancement of the acid groups of biomolecules accompanying the histochemical detection of sodium cations in the pulmonary and tracheal cell and tissue ultrastructures.

5 dwg, 1 ex

 

The invention relates to the field of medicine and biology, histological, cytological, physiological study of tissues and cells of human organs and animals, as well as by cytological and histological methods.

Known methods histochemical staining of sodium chloride tissues and cells bodies, which is carried out using a saturated aqueous solution hexahydroquinoline potassium (K[Sb(OH)6]), a 2% aqueous solution of OsO4/G. Geier. E-histochemistry. - M.: Mir. - 1974. - 488 p/. Methods of staining cells with an aqueous solution of antimonate and osmium acid required for the study of biological objects with an electron microscope, since the products of biological staining structures containing sodium ions, these methods become electron-dense.

The excess of osmium tetroxide prevents this histochemical reaction, as phospholipids containing polyunsaturated fatty acids, have high electron density. Additional contrasting nucleic acids contained in the nucleus and cytoplasm of the cells uranylacetate, citrate of lead, etc., as well prevents histochemical detection of sodium ions through antimonate. Therefore, the main drawback of these methods is that they are made without with�of NASA coordination compounds OsO 4(osmium acid) with hexahydrocannabinol potassium, which occurs during heating of the aqueous solution of these substances in a water bath and subsequent purification dissolved when heated coordination compounds OsO4in water, acetone excess of osmium tetroxide, which is poorly soluble in water and in organic solvents.

Loved ones are essentially ways histochemical detection of sodium chloride and calcium ions in cells and tissues, which are made using antimonate /Komnik H. Electron mikroscopische Lokalisation von Na+ and CI - in Zellen and Geweben // Protoplasma. - 1962. - V. 55. - P. 414-418. Mariani P., Tolomio C, Baldan B., and P. Braghetta Cell wall ultrastructure and cation localization in some benthic marine algae // Phycologia. - 1990. - Vol.29. - No. 2. - P. 253-262/.

The significant difference of the claimed method from this is that it is the synthesis and purification: 1) main coordination compounds of osmium tetroxide, which are required for histochemical staining of cells and tissues, and 2) K2CO3that required to neutralize dye solution. Is used for this in eightfold molar ratio to the osmium acid 0.1 M solution hexahydroquinoline potassium (K[Sb(OH)6]). While there is no osmiophilic cytoplasmic membranes, if the duration of the staining for 4 hours or more.

Similar technical results�tattoo are ways histochemical staining of fixed coordination compounds of osmium tetroxide with timelinelite, dimethylaminoacetyl, unbalanced dimethylethylenediamine, tetramethylethylenediamine, tetraethylethylenediamine, which are used for histochemical detection of substances with an acid reaction (nuclear chromatin, nucleolus, ribosomes, glycosaminoglycans, etc.) in tissues and cells /Geier G. E histochemistry. - M. Mir. 1974. - 488 p/.

The significant difference of the claimed method from this is that as the ligand is used a saturated aqueous solution hexahydroquinoline potassium (K[Sb(OH)6]), resulting in synthesized Os[Sb(OH)6]4and K2CO3and what for histochemical detection of sodium chloride, calcium ions, inorganic ions of the iron used K[Sb(OH)6].

Close according to an embodiment is a method of producing pyroantimonate mercury (II) /Servinski A. E. EN 2036149 C1/, which is used in inorganic chemistry in the formation of compounds of antimony and mercury. The inventive method: the solutions of acetate of mercury (II) and potassium hydroxide is added to a solution hexahydroquinoline potassium. The molar ratio of K[Sb(OH)6]:Hg(CH3COO)2:KOH=2:(2-3):(6-8). Was stirred and heated for 10-15 min. the Precipitate was separated, washed with hot water and air dried at 100-120°C. the drying Time of 15-60 min. the Essential difference of the claimed method from this is �the formation of the coordination compounds of osmium tetroxide - Os[Sb(OH)6]4and K2CO3finally formed in the presence of NaCl and sodium ions (Na+), calcium (Ca+) found in the investigated organs and tissues of the body.

The task of the claimed method is the additional okrashivanie nucleic acids in the nucleus and cytoplasm, which is performed by electroncapture contrasting cells required for histochemical detection of sodium cations in ultrastructure.

The essence of the claimed method is that using a transmission electron microscope are studied in ultrathin sections of tissues and cells, fixed in histological fixative for 1 hour, contains 2% aqueous solution hexahydroquinoline potassium, and then with the aim histochemical detection of phosphate groups contained in nucleic acids, are painted with an aqueous solution of the basic coordination compounds of osmium tetroxide - Os[Sb(OH)6]4containing K2CO3that finally formed in the presence of NaCl and sodium ions (Na+), calcium (Ca+) found in the investigated organs and tissues of the body. The reaction of histochemical staining occurs at a molar ratio of the reactants inside the pieces of tissues containing sodium chloride etc. and reagents in the incubation with�food K[Sb(OH) 6]:OsO4that is equal to 1:(1-8):(1-10).

Our proposed method involves the synthesis of coordination compounds of the main interaction of osmium OsO4and osmium acid with hexahydrocannabinol potassium, which finally occurs in tissues, organs, in cells which contains in access of atmospheric air CO2that is absorbed in K2O. After fixation, slices of organs in histological fixative containing hexahydrocannabinol potassium, is the staining of the slices of the main bodies of the coordination compound of osmium, washing in distilled water, dehydrated in alcohol or acetone, opinion pieces in Araldite-EPON, fabrication of ultrathin sections, the study of tissues and cells using transmission electron microscope, computer processing of electronography.

The invention is illustrated: Fig. No. 1 - Chondrocytes fibro-cartilaginous membrane of the trachea of the rat. In the cytoplasm of chondrocytes revealed acidic groups of glycosoaminoglycans. Colour is the principal coordination compound of osmium, Fig.2 - Respiratory Department of lungs of the rat. Cellular elements of the interalveolar septum, selective staining of heterochromatin, euchromatin, nucleolus, perichromatin, interchromatin pellets, the main coordinating compound of osmium, Fi�.3 - The wall of the alveoli of the lungs of rats. In'veolocity detected electron-dense granules of calcium dihydrogen phosphate. In the intercellular space of the deposition of diffuse electroncapture material of sodium dihydrogen phosphate, Fig.4 - Intense contrast enhancement of the coordination compound of osmium cytoplasm and nuclei of the connective tissue cells of the interalveolar septum. In the intercellular space of the observed fine-grained precipitate antimonate sodium, Fig.5 - Deposition of sodium dihydrogen phosphate surfactant in the contingency. Ultrathin section of the lungs of rats.

The method is carried out in three stages, following each other:

1. At the beginning of the method to a 2% aqueous solution of OsO4in an eightfold molar ratio of added saturated 2% (0.1 M) aqueous solution hexahydroquinoline potassium K[Sb(OH)6]. Pre-mix the solutions individually for 30 minutes and heated in a water bath at 100°C. Then the solutions are mixed, and then gradually cooled with constant vigorous shaking to ensure access of atmospheric air rich in CO2that is absorbed in K2O.

2. For the purpose of chemical coagulation of the proteins, small (5 mm thick, 5 mm long) pieces of tissue of human or animal are recorded in histological fixative (glutaraldehyde) for 1 hour, then at�OSU 2% aqueous solution hexahydroquinoline potassium adjusted values of a latch to a pH of 7.4 (added dropwise). After that, the surface of pieces of bodies quickly washed from the first latch in bidistilled water and then in 2% aqueous solution hexahydroquinoline potassium. Then for 4 hours or more, pieces of tissues are stained in 4% solution of the basic coordination compounds of osmium and K2CO3bidistilled water. Staining is performed with constant vigorous shaking to ensure access of atmospheric air rich in CO2that is absorbed in K2O. After staining the tissue pieces prepariruetsya by cutting on a thin (1-2 mm thick, 2-2. 5 mm long), then rinsed in bidistilled water, dehydrated in ethyl alcohol or acetone and poured into araldit-araldit.

3. At this stage, on the instrument are made of ultra-thin tissue sections, which are studied using transmission electron microscope with subsequent computer processing of electronography with 3D graphics.

An example of use.

The proposed method further electroncapture contrasting acidic groups in biomolecules in histochemical detection of sodium cations in ultrastructure cells and tissues was used for histochemical studies of the respiratory Department of lungs and trachea intact rats. Malen�Chia (5 mm thick and 10 mm long) pieces of the tissues of the lungs and trachea were fixed for 1 hour in 4% solution of glutaraldehyde, to which was added dropwise 2% hexahydrocannabinol potassium in order to achieve a physiological pH of a solution, equal to 7.4. Quickly washed first in bidistilled water and then in 2% aqueous solution hexahydroquinoline potassium. Two slices (5 mm thick and 5 mm long) was placed in a reagent solution, which contained 1 ml of 4% osmium tetroxide and 8 ml of 2% hexahydrocannabinol potassium. The volume of pieces of organs empirically determined that the concentration of sodium chloride and cations of sodium, calcium, etc. in the body is approximately equal to 0.1 M. for 4 hours the pieces of tissues stained with vigorous shaking in 2% solution of the basic coordination compounds of osmium with hexahydroquinoline potassium bidistilled water. After staining, the slices quickly rinsed in bidistilled water. Then the tissue pieces prepariruetsya by cutting on a thin (1-2 mm thick, 2-2. 5 mm long). For the purpose of dewatering the tissue pieces are held through a series of alcohols, acetone and then poured into Araldite-EPON. Then on the ultramicrotome LKB NOVA were made of semi-thin and ultrathin sections.

In the case study of semi-thin sections of fibrous (fibrous)-cartilaginous membrane of the trachea and lungs is detected by the high-intensity granular staining of the cytoplasm of chondrocytes and chondrogenesis�lastow, cells of the alveoli and interalveolar septa, which is rich in glycosaminoglycans (Fig.1).

In the study of ultrathin sections of the lungs with a transmission electron microscope FEI Tecnai G2 Spirit was revealed diffuse selective staining of acidic cellular ultrastructure and intercellular substance, and the lack osmiofilii cytoplasmic membranes. In the nuclei of cells detected by selective staining of heterochromatin, euchromatin, nucleolus, perichromatin, interchromatin granules, which are characterized by high content of ions of phosphoric acid

In the cytoplasm of cells of the alveoli is detected weakly colored or moderately electronmobility diffuse material, faint granular material of 0.01-0.05 μm and a single electronmobility granules of calcium dihydroorotate Ca(H2PO4)2the size of 0.09-0.3 mm (Fig.3). In the cytoplasm of erythrocytes contained in the capillaries of the lungs, observed weak staining of the cytoplasm, found very small electron-dense granules of size 0.01-0.02 μm (Fig.2).

The conclusion about the presence of inclusions of calcium dihydroorotate Ca(H2PO4)2in lung cells and red blood cells made on the basis of previously conducted histochemical studies of the lungs of rats, which was carried on with�OSU alizarin red C /S. V. Zinoviev Histochemical characteristics of the venous system of the respiratory Department of lungs of experimental animals subjected to chronic hypothermia, after introduction into the body of dihydroquercetin // Bulletin of physiology pathology of respiration. - No. 45. S. 57-61/. In the intercellular substance in the cytoplasm and wall aeroheating barrier detects accumulation of neutral lipids, which are adjacent to diffuse accumulations electroncapture material size of 1-4 μm (Fig.2, 3).

On the surface of'veolocity first type is present diffusely painted a layer of surfactant which has a low electron density. In the surfactant varies two layers of gipofiz. On the surface of the surfactant are single very fine granules osmiophilic material (Fig.2, 3).

In the alveoli found colored coordination compound of osmium material, disordered located between subcellular structures in the intercellular substance in the contingency surfactant, in accumulations of neutral lipids, which contains molecules of sodium dehydroacetate (NaH2PO4) with moderate electron density, represented by a granule size of from 0.05 to 0.3 microns. The edges of these granules blurred, indistinct contours, some of them have a round or star-shaped (Fig.5).

Software Tr�nsmission electron microscope FEI Tecnai G2 Spirit allows you to carry out computer processing of electronography. When preprocessing electronography 3D graphics found crystals precipitate of antimonate, which contain sodium chloride and has a complex structure. With increasing electron microscope over 30,000 times, in the case of computer processing of electronography observed that the composition of the cytoplasmic vacuoles and in the intercellular space are detected fine-grained precipitate hexahydroquinoline potassium (0,0001-0,005 µm) with low or moderate electron density, which is formed by interaction with the sodium chloride (Fig.3, 4).

The method allows to synthesize, purify acetone basic coordination compounds of osmium - Os[Sb(OH)6]4excess of osmium tetroxide, which are used for histochemical detection of nucleic acids in the presence of NaCl and sodium ions (Na+), calcium (Ca+) found in the investigated organs and tissues of the body.

The technical result of the use of the method is that there is a qualitative staining of tissues, cells by inhibition of staining by osmium tetroxide phospholipids, with the aim of identifying nucleic acids (DNA and RNA), as well as sodium chloride solution, the cations of calcium, iron, etc., allowing you to spend significant ultrastructural study of ultrathin sections using Tr�nsmission electron microscope.

Thus, our proposed method can be used for electron microscopic studies of ultrastructure of cells and intercellular substance of organs and tissues of humans and animals, which have an acidic pH environment, Obnovlenie the presence of ions of phosphoric acid, sulfuric acid, and molecules of sodium chloride, or sodium cations.

The method further electroncapture contrasting acidic groups of the biomolecules in the histochemical detection of sodium cations in ultrastructure cells and tissues of the lungs and trachea, characterized by the fact that the pieces of 5 mm thick and 10 mm long tissue of the lung and trachea is fixed for 1 hour in 4% solution of glutaraldehyde, which was added dropwise 2% hexahydrocannabinol potassium to achieve a pH of 7.4, carry out cleaning of the surface from the retainer in bidistilled water and then in 2% aqueous solution hexahydroquinoline potassium, slices of 5 mm thickness and 5 mm in length is placed in a reagent solution,which contains 1 ml of 4% osmium tetroxide and 8 ml of 2% hexahydroquinoline potassium, for 4 hours, the tissue slices were stained with vigorous shaking in 2% solution of the basic coordination compounds of osmium with hexahydrocannabinol potassium bidistilled water, rinsed in bidistilled water, then the slices of tissue preparer�Ute by cutting on thinner 1-2 mm thickness 2-2,5 mm long, dehydrate, carried out through a series of alcohols, acetone and then poured into Araldite-EPON, and then on the instrument are made of semi-thin and ultrathin sections, which are examined with a transmission electron microscope, with subsequent computer processing of electronography 3D graphics with the detection of diffuse selective staining of acidic cellular ultrastructure and intercellular substance.



 

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3 cl, 4 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to method of obtaining 1,6-bis-(1,5,3-dithiazepan-3-yl)-2,5-disulphanylhexane of formula (1), possessing fungicidal activity against Botrytis cinerea and Rhizoctonia solani. Essence of method lies in interaction of mixture of 1,2-ethanedithiol and formaldehyde with water solution of ammonium salts NH4X (X=F, Br, OAc, NO3, 1/2SO4) with molar ratio HS(CH2)2SH:CH2O:NH4X=3:6:2 at room temperature (~20°C) and atmospheric pressure for 5-7 h.

EFFECT: output of 1,6-bis-(1,5,3-dithiazepan-3-yl)-2,5-disulphanylhexane of general formula (1) depending on applied ammonium salts (NH4X) constitutes 31-67%.

2 cl, 2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: paleontological objects with soft tissues are treated for 1-4 months with an embalming solution containing 40% formalin - 0.516%, crystalline phenol - 0.262%, glycerine - 79.077%, 96% alcohol - 18.576%, sodium chloride - 1.569%. The ratio of the embalming solution to the mass of the paleontological object must not be less than 3:1. Embalming is carried out at room temperature.

EFFECT: method of embalming paleontological objects with soft tissues provides prolonged preservation of soft tissues, minimises loss of soft tissues having different rotting stages, stops rotting processes, skin straightening, preserves natural colour thereof and natural morphometric parameters, which enables further use of the paleontological object for scientific purposes and for exhibition in dry form.

3 cl

FIELD: medicine.

SUBSTANCE: invention refers to physiology, cryobiology and medicine, namely to human blood nuclear cell preservation technique with the use of inert gas. The method involves cell saturation in Compoplast 300 plasticised container placed in a metal cryobarocontainer with inert gas, xenon at pressure 0.6 atm for 20min in the room temperature environment of 21±2°C; excessive gas is eliminated, and the biological object is removed from the metal container and frozen to -28°C in ethanol, placed into an electric freezer at -80°C. The bioobject is unfrozen in a water bath at 39±1.5°C for 1.5 min.

EFFECT: implementing the invention provides the reliable cryonic preservation of leukocytes in the inert gas medium and the high level of qualitative and morphological safety of leukocyte concentrate of human blood.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method of reducing impact of low-temperature jump on cryoprotectant solution is provided at the expense of remote processing of cryoprotectant solution with cells of living organisms by ultrasonic radiation with frequency 0.50-10 before its freezing.

EFFECT: reduction of low-temperature jump of cryoprotectant solutions, which makes it possible to eliminate overcooling effect, provide continuous and smooth character of freezing and increase integrity of defrosted cells after cryoconservation.

1 dwg

FIELD: medicine.

SUBSTANCE: what is presented for embalming of dead bodies for the purpose of organ harvesting and transplantation drills is a method, wherein tissues and organs are saturated with embalming solutions; the dead body is immersed into water at t 40°C for 120-160 min; cannulas are inserted into a femoral artery, and a blood flow is washed with warm normal saline with 1-2% sodium citrate added; the femoral artery is sealed, and a solution containing formalin and glycerol is inserted into the artery.

EFFECT: keeping the internal organ structure by increasing the preserving properties of the solution and providing the small vessel filling.

FIELD: chemistry.

SUBSTANCE: invention relates to a method of lyophilisation of a composition, which contains purified antithrombin III (AT III) and a crystallised substance, selected from alanine, mannitol, glycine or NaCl. The claimed method includes freezing the composition at a temperature from -52°C to -60°C for 6-15 hours, annealing the composition at -30°C for 1 hour, re-freezing the composition at a temperature from -52°C to -60°C for 2-15 hours at keeping the product temperature between -48°C and -52.7°C for 4-10 before lyophilisation and drying the composition with obtaining a lyophilised cake. The invention also relates to a pharmaceutical set, which contains the said lyophilised cake and a liquid reagent.

EFFECT: invention provides obtaining the lyophilised composition, containing AT III, which preserves its activity and stability.

14 cl, 24 dwg, 5 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl

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