RU2647420C1 - Combined method of hystochemical detection of dezoxyribonucleoprotenins, polysaccharides and hydrocarbon components of bio polymers in semitinous sections of tissues and organs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of experimental medicine and biology, in particular to histological studies, and is intended for microscopic studies of semitinous sections of organs and tissues filled in a mixture of epona and araldite. For this purpose, the fabricated semitinous sections are subjected to alkaline hydrolysis with 0.5–0.7 M solution of potassium hydroxide in absolute methanol for 10–15 minutes. Samples are washed three times for 3 minutes with absolute ethanol, and then with distilled water. Then samples are dried in air at room temperature and then incubated for 8 minutes with a 1 % aqueous solution of potassium periodate, followed by rinsing with distilled water. Samples are air dried and incubated with Schiff's reagent for 30 minutes in the dark, after which they are washed for 2–3 minutes with running water and then with sulfurous and distilled water.

EFFECT: invention makes it possible to detect deoxyribonucleoproteins, polysaccharides and carbohydrate components in semitinous sections of various organs without heating and damage to the sections.

1 cl, 7 dwg, 4 ex

Description

The invention relates to experimental medicine and biology, namely to histological studies. The most commonly used histochemical reactions for light microscopy are the Felgen reaction and the SIR reaction (Schiff-iodic acid reagent) [3]. The SIC reaction reveals polysaccharides and carbohydrate components of glycoproteins in the slices and consists in the oxidation of polysaccharides with potassium or sodium periodate to form aldehyde groups from the hydroxyl groups, which give a reaction with fuchsulfuric acid (Schiff's reagent) with the formation of raspberry colored products [4]. The Felgen reaction reveals pentoses in the composition of deoxyribonucleoproteins and is also based on the use of Schiff's reagent. Its peculiarity is preliminary acid hydrolysis of nucleoproteins at a temperature of 60 ° C instead of using periodate [4]. During acid hydrolysis in deoxyribonucleic acid (DNA), glycosidic bonds are broken that connect the pentose to the nitrogenous base, as a result of which reactive aldehyde groups at the first carbon atom are released in pentoses, which then react with the Schiff reagent. Many polymers containing carbohydrates are hydrolyzed, depolymerized and extracted, as a result of which the positive Felgen reaction and the SIR reaction cannot occur simultaneously, and their joint implementation is impractical due to the high probability of false negative results.

Of particular difficulty is the histochemical detection of deoxyribonucleoproteins and carbohydrate structures in semi-thin sections of tissues and organs embedded in resins for electron microscopy. The manufacture of semi-thin sections with a thickness of 1-2 μm precedes the electron microscopic examination of biological preparations and is necessary to identify cells and non-cellular structures of interest to the researcher, for the subsequent manufacture of ultra-thin sections. Histochemical methods that are widely used in the study of histological preparations made from pieces of tissue dehydrated in alcohols and embedded in paraffin do not always reproduce on semi-thin sections, since when preparing the tissue for electron microscopy, the samples are contrasted with osmium oxide, uranyl acetate, which significantly affects the course of histochemical reactions, and then poured into epoxy resins, which, when polymerized, change the properties of biopolymers [1]. Conducting histochemical reactions aimed at identifying the main biopolymers in cells, such as nucleic acids, polysaccharides and glycoproteins, would significantly increase the accuracy of detection of organelles and inclusions, as well as the differentiation of structures similar in structure, such as fragmented nuclei, secretory granules, lysosomes, which is significantly would increase the accuracy of the selection of tissue and organ sections for electron microscopy, eliminate the need to fill and study more objects, and accordingly Twain, reduced the cost of the study. In addition, these techniques would significantly expand the use of semi-thin sections in histological and cytological studies using light microscopy, since semi-thin sections allow visualization of structures inaccessible to the study of classical histological preparations. The use of histochemical reactions, the identification and differentiation of intracellular formations could in some cases be carried out without the use of electron microscopy, which also significantly reduces the cost of the study.

The Felgen reaction on semi-thin sections is difficult, since resins and contrast agents inhibit the manifestation of color [2]. The closest analogue of the Felgen reaction in semi-thin sections is the technique developed for non-contrasted preparations [9]. Samples for research are fixed in glutaraldehyde without postfixation with osmium oxide and poured into Epon. Semi-thin sections of 1-2 microns thick are made. Acid hydrolysis is carried out for 1 h at room temperature in 5 N. hydrochloric acid solution. Then Schiff's reagent is applied to the sections and incubated in the dark for 1-2 hours. The nuclei are painted in bright pink.

The disadvantage of this method is to obtain a similar stain without acid hydrolysis, which is a determining factor in the development of the Felgen reaction, only due to the reaction of the Schiff reagent with glutaraldehyde present in the sample, that is, staining is a nonspecific reaction, which was established by the developer of the method [9] . In addition, sample preparation does not allow their further use for electron microscopic studies.

Several methods are known for conducting a SEC reaction on semi-thin sections [5, 6]. Their common disadvantages are the strong damaging effect on the fabric of additional oxidizing agents, such as hydrogen peroxide, as well as the ability to give specific staining of drugs only when using a certain type of filling resin (methacrylate, Epon, Araldit). Currently, as a rule, a combined mixture of Epon and Araldit resins is used for the manufacture of drugs, and methacrylate is practically not used in electron microscopy.

The closest analogue to the claimed method is the method proposed by R. DiBella and K. Hashimoto [7], to conduct the SEC reaction in semi-thin sections. A few drops of 0.5% potassium periodate are applied to the preparations of biological samples poured into Araldit for 10 minutes and the glasses are heated on a heating table to 65 ° C. To avoid evaporation, periodate is added to the slices as needed. Then the glass is immersed in distilled water, and then heated again until the water evaporates. Schiff's reagent is applied dropwise to a preparation warmed up to 65 ° C for 5 minutes, periodically adding to prevent evaporation. The reagent changes color to purple, after which the sections are washed in distilled water.

The disadvantage of this method is the need for repeated incubation of sections at elevated temperature, which causes the drug to dry out, and then, when washing in distilled water, peeling and loss of sections. The method does not stain biological samples embedded in other casting resins. It is not combined with other histochemical methods.

The objective of the invention is to develop a technique that allows simultaneous histochemical detection of DNA, polysaccharides and carbohydrate components of various classes of biopolymers (glycoproteins) in semi-thin sections of samples poured into combined resins, giving specific staining of various tissues and organs, with minimal damaging effects on the sections.

The claimed method for simultaneous histochemical detection of DNA, polysaccharides and carbohydrate components of biopolymers in semi-thin sections of biological samples embedded in a mixture of epon and araldite resins consists in immersing the sections in a 0.5 M solution of potassium hydroxide in absolute methanol for 10 min at room temperature, transferring the sections in the first absolute ethanol for 3 minutes, then in the second and third absolute ethanol also with an incubation of 3 minutes. Then the sections are washed in three shifts of distilled water for 3 minutes. After the third washing, excess water is carefully removed from the glasses around the sections with filter paper, and the slides with sections are left to dry at room temperature for 3-4 minutes. Then, a 1% aqueous solution of potassium periodate is added dropwise to the sections and incubated at room temperature for 8 minutes. Immerse the glass in distilled water for a few seconds to remove the periodate. Then, excess water is removed from the glasses around the sections with filter paper. Slices are dried at room temperature for 3-4 minutes. Schiff's reagent is applied dropwise to the sections and the sections are placed in the dark for 30 minutes. After incubation, the sections are immersed for 2-3 minutes in running cold water. At this time, sulphurous water is prepared by adding 0.5 g of sodium metabisulfite and 5-6 drops of 1N to 100 ml of distilled water. of hydrochloric acid. Then it is transferred for 1 min into sulphurous water, after which it is washed in distilled water for 1 min. Remove excess water from the glasses with filter paper, leave the slices at room temperature for 5 minutes to dry, then apply the medium for the conclusion of the preparations and cover with a coverslip.

The technical result consists in the appearance of stable specific staining of structures containing DNA and carbohydrates in shades from pink-lilac to raspberry-violet in various organs and tissues embedded in a mixture of Epon and Araldite, without loss and thermal damage to semi-thin sections.

The specified technical result is achieved by the fact that in the proposed method, acid hydrolysis of DNA at an elevated temperature is replaced by treatment with an alcohol solution of alkali. To identify DNA in semi-thin sections of the samples, it was necessary to make DNA pentoses available for the SHIC reaction, as with other polysaccharides and glycoproteins, that is, to free their hydroxyl groups for reaction with periodate, which will avoid breaking glycosidic bonds and oxidation of pentoses with acid. To form reactive aldehyde groups in pentoses, alkaline hydrolysis was carried out in a 0.5 M solution of potassium hydroxide in methanol. The action of alkali causes the removal of phosphoric acid residues from nucleotides to form nucleosides. There is no rupture of glycosidic bonds, but the hydroxyl group at the third carbon atom of pentose is released. This makes it possible to oxidize it with periodate, and then carry out the reaction with Schiff's reagent. The choice of absolute methanol as a solvent is due to the following: alcohols facilitate the penetration of the reagent into sections, but during the preparation of a water – alcohol solution, the Canizzaro reaction can occur with the formation of alcohols and acids from carbohydrates [4], which may interfere with DNA detection. Therefore, absolute alcohol was chosen. The choice of methanol is due to the fact that it is not able to react with alkalis, but lowers the pH of the solution, which is important for the preservation of biopolymers. At the same time, it also makes it possible to increase the reactivity of carbohydrates in DNA and in other complex compounds by dissolving resins, as well as to facilitate the interaction of potassium periodate with carbohydrate hydroxyls. It is known that some polysaccharides, for example, glycosaminoglycans, react poorly with periodate, since their carboxyl and sulfate groups cause repulsion of the periodate ion, as a result of which the SIR reaction may not reveal them [8]. The treatment of slices with alkali was to minimize these phenomena. To increase the specificity after alkaline hydrolysis, multiple washing of the drug was used, first in polar (absolute ethanol), then in non-polar solvents (distilled water) to remove organic and inorganic hydrophobic and hydrophilic compounds, including the fixative, since the SIR reaction can give false positive results on biological samples fixed with glutaraldehyde (the most common fixative for electron microscopic studies), since it is believed that of free aldehyde groups may react with Schiff's reagent. The potassium periodate concentration was increased from 0.5 to 1% to increase the oxidation efficiency at room temperature. The enhancement of the reaction with the Schiff reagent due to an increase in the temperature of the reaction medium was replaced by an increase in the duration of incubation with the Schiff reagent at room temperature.

To evaluate the effectiveness of the claimed method conducted the following studies.

Example 1. Determination of the specificity of developing color

To establish the specificity of the simultaneous determination of carbohydrate components and DNA, histochemical reactions were performed by the claimed method in samples of organs having biological structures that should give a positive reaction to carbohydrate components and DNA, as well as a negative reaction to DNA and carbohydrates. The object of the study was the semi-thin sections of the thyroid gland and blood vessels. Thyroid preparations are a positive test for the CHIC reaction due to the content in the follicle cavities of a large amount of colloid - a highly glycosylated thyroglobulin protein. Also, a positive CHIC reaction is provided by the basement membranes of follicular thyrocytes and capillaries. At the same time, the nuclei of thyroid cells give a positive Felgen reaction. Blood plasma in the lumen of blood vessels contains a large number of glycoproteins, giving a SIC-positive reaction. The erythrocytes do not have nuclei and do not contain a large amount of carbohydrates in the cytoplasm, as a result of which they do not give either the SIR reaction or the Felgen reaction, i.e. they are a negative control.

The material was fixed with 1% glutaraldehyde with postfixation with a 1% solution of osmium tetrooxide in cacodilate buffer and a 5% aqueous solution of uranyl acetate and poured into a mixture of Epon and Araldit with the addition of dibutyl phthalate as a plasticizer and dodecyl succinic anhydride DMP30 as a catalyst. We stained the manufactured semi-thin sections according to the claimed methodology. In FIG. 1 shows the results of histochemical reactions in the thyroid gland. The colloid in the lumen of the follicles is colored raspberry-violet, the nuclei of thyrocytes are lilac-pink with a more intense color of the nucleoli, the cytoplasm of the cells is not stained, in the cytoplasm you can see the SHIK-positive inclusions of thyroglobulin (indicated by arrows), in the lumen of blood vessels unpainted erythrocytes. In FIG. 2 presents the results of staining sections of the vessel, in the form of a plasma painted in pink and crimson in the lumen of the vessel and unpainted red blood cells (indicated by arrows). The safety of the slices was one hundred percent.

Thus, the claimed method is characterized by efficiency in the formulation of the reaction on samples embedded in combined epoxy resins, does not give false positive and false negative reactions and, therefore, is a specific method for identifying carbohydrate components and DNA.

Example 2. Determining the sensitivity of detection of DNA and carbohydrate component

The brightness of the developing color of the nuclei depends on the amount of heterochromatin, which has a high compaction of nucleoproteins, and more loose euchromatin, respectively, giving a weaker color of deoxyribonucleoproteins. Similarly, the color saturation of carbohydrate components depends on their quantity in the studied object, which makes it possible, if necessary, for spectrophotometric determination of the amount of these compounds. To determine the sensitivity of the claimed method, histochemical detection of glycoproteins and DNA in the thyroid gland in follicles with different functional state of the cells was performed. Fixation and filling of the material was carried out in a similar manner as in example 1.

In FIG. Figure 3 presents the results of the detection of thyroglobulin in the group of thyroid follicles with different contents of this glycoprotein with a corresponding coloration of the contents of the follicle cavities from light pink-lilac to saturated raspberry color. Euchromatin of nuclei gives a weak positive reaction, heterochromatin along the nuclear membrane and nucleoli stain more intensely. Cell cytoplasm does not stain. A positive SIC reaction is observed in the basement membranes of follicular thyrocytes.

In FIG. 4 presents the results of a histochemical reaction in thyroid cells with different nuclear states. The pycnotized nuclei of dying cells desquamated into the cavity of the follicles (indicated by arrows) give a more intense color due to a higher concentration of nucleoproteins.

Thus, the claimed method of histochemical detection is sensitive.

Example 3. The identification of polysaccharides that impede the course of the SIC reaction

Carbohydrates, in the structure of which there are groups that prevent the formation of aldehyde groups in carbohydrates, include glycosaminoglycans. To determine the effectiveness of the claimed method, histochemical detection of mast cell heparin, proteoglycan, consisting of protein and glycosaminoglycans, was performed. To avoid misinterpretation of negative results, the reaction was carried out as described above on semi-thin sections giving a positive SHIK reaction of the thyroid gland, in which connective tissue septa there are often mast cells.

In FIG. Figure 5 shows the results in the form of a sharply positive reaction with the development of a saturated raspberry-violet color of the granules in the cytoplasm of mast cells, bright pink-purple staining of the colloid in the follicles, and raspberry-violet staining of heterochromatin and nucleoli of follicular thyrocyte nuclei.

Thus, the claimed method allows the detection of glycosaminoglycans, which increases the applicability of the SIK reaction.

Example 4. The lack of organ specificity of the claimed method

To establish the effectiveness of the claimed method, DNA and carbohydrates were detected in various organs. Preparations of the cortical substance of the adrenal glands and testes of rats were made as described above in example 1. The cortical substance of the adrenal glands is represented by cells whose cytoplasm contains a large number of large oval-shaped mitochondria and single lipid droplets that do not give a positive reaction with Schiff's reagent. Cells are surrounded by capillaries, whose basement membrane is CHIC-positive. In the testes, the spermatogenous epithelium contains large nuclei with decondensed chromatin of low density, giving a weak Felgen reaction. Sperm cells contain highly condensed chromatin in the head, and a large amount of glycoproteins on the plasmolemma in the tail, resulting in a sharply positive SIR reaction and the Felgen reaction.

In FIG. Figure 6 shows the results of the reaction on the adrenal cortex. The cell nuclei have a lilac-pink color, large unpainted lipid drops (indicated by arrows) and smaller unpainted mitochondria are visible in the cytoplasm. The basement membranes of the capillaries give a bright color.

In FIG. 7 presents the results of histochemical reactions of the claimed method in the preparations of the testes. In spermatogenous epithelium, pink-lilac color gives chromosomes, bright raspberry-violet color - heads, and raspberry-lilac color - tails of sperm (indicated by arrows).

Therefore, the claimed method is characterized by the absence of organ-specificity of positive reactions, that is, it is universal for detecting DNA and carbohydrate components in organs and tissues.

Thus, the claimed method is an effective method for detecting DNA, polysaccharides and carbohydrate components of biopolymers in semi-thin sections of samples of various tissues and organs embedded in a mixture of various epoxy resins. This method is also characterized by high material safety and ease of execution, since it does not require the use of thermostats and heating tables.

Literature

1. Gayer G. Electronic histochemistry. - M .: Mir, 1974. - 488 p.

2. Konarev V.G., Tyuterev S.L. Methods of biochemistry and cytochemistry of plant nucleic acids. - L .: Kolos, 1970 .-- 204 p.

3. Petrova A.S., Polonskaya N.Yu. Chapter 17. The main methods of staining of cytological preparations // Microscopic technique: manual / Ed. D.S. Sarkisova, L.Yu. Perova. - M.: Medicine, 1996 .-- 544 p.

4. Pierce E. Histochemistry. Per. from English - M .: Publishing house of foreign literature, 1962. - 962 p.

5. Carson F., Hladik C. Histotechnology: A Self-Instructional Text (3 ed.). - Hong Kong: American Society for Clinical Pathology Press, 2009 .-- P. 137-139.

6. Cerri PS, Sasso-Cerri E. Staining methods applied to glycol methacrylate embedded tissue sections // Micron - 2003. - Vol. 34. - No. 8. - P. 365-372.

7. DiBella R., Hashimoto K. A new method for PAS stain of osmium-fixed Araldite-embedded thick tissue section // J. Invest. Dermatol. - 1966. - Vol. 47. - P. 503-505.

8. Scott J., Harbinson R. Periodate oxidation of acid polysaccharides. II. Rates of oxidation of uronic acids in hyalyuronides and acid mucopolysaccharides // Histochemie. - 1969. - Vol. 19. - P. 155-161.

9. Stockert J. Feulgen positive nucleoli in Epon semithin sections: Fact or artifact? // Experientia. 1978. - Vol. 33. - No. 12 .-- P. 1669-1670.

Claims (1)

A method for simultaneous histochemical detection of deoxyribonucleoproteins, polysaccharides and carbohydrate components of biopolymers in semi-thin sections of tissues and organs, which consists in oxidizing the sections with a 1% aqueous potassium periodate solution for 7-10 minutes, followed by washing the sections in distilled water and drying the sections; incubation in the dark with Schiff's reagent for 25-40 minutes, followed by washing for 2-3 minutes in flowing, 1 minute in sulphurous and distilled water and obtaining a pink-violet color in the samples, characterized in that the samples pre-filled in a mixture of epon and araldite is hydrolyzed with a 0.5-0.7 M solution of potassium hydroxide in absolute methanol at room temperature, followed by removal of the hydrolysis products by washing with absolute ethanol and distilled water.

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