New application of dimethyl carbonate and method for preparing animal and plant tissue paraffin section by using dimethyl carbonate as transparent reagent

Abstract

The invention discloses a new application of dimethyl carbonate and a method for preparing animal and plant tissue paraffin sections by using the dimethyl carbonate as a transparent reagent, and belongs to the technical field of biological sections. The invention discloses a new application of dimethyl carbonate. The dimethyl carbonate is used as a transparent reagent for preparing animal and plant tissue slices. The dimethyl carbonate used in the invention has the characteristics of no pungent smell, no toxicity, no carcinogenicity, miscibility with other organic reagents, biodegradability and the like. Toxicological experiment analysis shows that dimethyl carbonate has no obvious influence on key organs of mice, does not induce obvious inflammatory response, and shows higher biological safety. And the obtained animal and plant tissue slice wax belt is smoother, good in continuity and not easy to break. The obtained animal and plant tissue sections are dyed more uniformly, the integrity of animal tissue structures can be kept, and the form and integrity of plant parenchyma cells can be well maintained.

Description

Technical Field

[0001] The invention belongs to the technical field of biological sections, and more specifically relates to a new use of dimethyl carbonate and a method for preparing paraffin sections of animal and plant tissues as a transparent reagent. Background Art

[0002] Paraffin sectioning is a basic and important technique in microscopic observation and histological research, and is widely used in pathology, cell biology, developmental biology, genetics and other fields [non-patent literature 1]. For example, in clinical diagnosis, it is mainly used to diagnose diseases, evaluate treatment effects and monitor disease progression; in basic research, it is mainly used to observe the morphological structure of cells and tissues, explore cell-to-cell interactions and signal transduction pathways. With the deepening of research, many emerging sectioning techniques such as ultrathin sections, semi-thin sections, and frozen sections have been developed based on paraffin sectioning. Although modern technologies such as frozen sections and laser microdissection provide new options, paraffin sectioning technology still occupies an important position in many fields due to its advantages such as low cost, easy operation, continuous sectioning, long-term preservation and uniform sectioning [non-patent literature 2].

[0003] The main technical principle of the paraffin section method is to transform biological tissues into observable slices through a series of delicate steps, so as to observe and study cell structure, tissue morphology and pathological changes under a microscope. Its basic steps include tissue fixation, dehydration, transparency, wax immersion, embedding, sectioning and staining. Among them, transparency is a key step in the paraffin section technology that connects the past and the future, and is a bridge between the dehydration and wax immersion steps, while the transparent agent plays an important role as a medium between the dehydrating agent and the penetrant. Relying on the property of being able to mix with both the dehydrating agent and paraffin, the transparent agent can quickly replace the dehydrating agent in the tissue cells. In the subsequent wax immersion step, the transparent agent can be completely replaced by paraffin, thereby realizing tissue wax immersion and embedding.

[0004] To date, the commonly used transparent reagent for paraffin sections is still xylene [Non-patent Document 3]. Although it has excellent penetrating power, it also has obvious disadvantages, such as making tissues and organs brittle and hardened. In addition, the reagent is toxic, has a pungent odor, is volatile at room temperature, can be absorbed through the respiratory tract, digestive tract and skin, and quickly enters the blood and is distributed to fat-rich tissues, with the highest concentrations in the brain, adrenal glands, bone marrow and liver. Inhaling a high concentration of this product in a short period of time can cause acute poisoning, and dry skin, chapped skin and dermatitis often occur after skin contact. Long-term contact can cause neurasthenia syndrome [Non-patent Document 4]. In addition, xylene is also toxic to marine and freshwater organisms when it spreads in the aquatic environment [Non-patent Document 5]. The U.S. Agency for Toxic Substances and Disease Registry (ATSDR / EPA) has listed it in the list of hazardous (toxic) air pollutants [Non-patent Document 6], and the World Health Organization's International Agency for Research on Cancer listed it in the list of Class 3 carcinogens in 2017. Therefore, it is very necessary to find a green, non-toxic and more environmentally friendly transparent reagent to replace xylene.

[0005] Since xylene was applied to tissue sections, researchers have been looking for its substitutes as clearing agents. Some studies have used chemical reagents such as n-butanol [non-patent document 7], isopropanol [non-patent document 8], n-heptane [non-patent document 9], trichloroethane [non-patent document 10], chloroform [non-patent document 11], D-limonene [non-patent document 12], propylene glycol methyl ether [non-patent document 13], and butyl decanoate [non-patent document 14] to replace xylene as a clearing agent; researchers have also used natural plant oils such as olive oil and coconut oil [non-patent document 15], wintergreen oil [non-patent document 16], and cedar oil [non-patent document 17] as substitutes. Mixtures such as isopropanol mineral oil mixtures [non-patent document 18], soap water mixtures [non-patent document 19], and mixtures of aliphatic hydrocarbons and d-limonene [non-patent document 20] have also been developed as substitutes. The above-mentioned clearing agent substitutes have been used to optimize and improve the paraffin sectioning methods of various animal and plant tissues, but the optimization results are not ideal, and the actual toxicological experiments and effects of replacing new reagents have not been reported. Among them, many chemical reagents are known to have higher toxicity than xylene; studies have found that solvents such as limonene, benzene, toluene, chloroform and petroleum ether can also damage tissue samples [Non-patent literature 21].

[0006] At present, there is no widely used biosafe, green and environmentally friendly transparent reagent that can replace xylene in the process of paraffin sectioning of animal and plant tissues.

[0007] Prior art literature [Non-patent literature 1]: Zhanmu et al., 2020; [Non-patent document 2]: Xu and Wei, 2020; [Non-patent document 3]: Rodig, 2021; [Non-patent document 4]: Niaz et al., 2015; [Non-patent document 5]: Duan et al., 2017; [Non-patent document 6]: Stockert et al., 2012; [Non-patent literature 7]: Cai et al., 2015; Wang et al., 2024; Zhanmu et al., 2020; Zhao et al., 2017; [Non-patent literature 8]: Falkeholm et al., 2001; Metgud et al., 2013; Viktorov and Proshin 2003; [Non-patent literature 9]: Stockert et al., 2012; Wang et al., 2024; [Non-patent document 10]: Maxwell, 1978; Temel et al., 2005; [Non-patent literature 11]: Zhanmu et al., 2020; [Non-patent document 12]: Longman et al., 2013; [Non-patent literature 13]: Chen et al., 2010; [Non-patent document 14]: Lyon et al., 1995; [Non-patent document 15]: Bruun Rasmussen et al. 1992; [Non-patent literature 16]: Koay et al., 2016; [Non-patent literature 17]: Indu et al., 2014; [Non-patent document 18]: Buesa and Peshkov 2009; [Non-patent literature 19]: Ankle and Joshi, 2011; Pandey et al., 2014; [Non-patent document 20]: Zhanmu et al., 2020; [Non-patent literature 21]: Birkner and Woltman 2007; Metgud et al., 2013. Summary of the invention

[0008] In order to overcome the above defects, the present invention provides a method for preparing paraffin sections of animal and plant tissues using dimethyl carbonate as a transparent reagent, which has the advantages of being green, non-toxic, and having little environmental pollution.

[0009] The object of the present invention is achieved by the following technical solutions: A new use of dimethyl carbonate is to use it as a transparent reagent for preparing animal and plant tissue sections.

[0010] Preferably, it is used as a transparent reagent for preparing paraffin sections of animal and plant tissues.

[0011] The present invention also provides a method for preparing paraffin sections of animal and plant tissues using dimethyl carbonate as a transparent reagent, comprising the following steps: S1 Sample fixation: Cut the fresh tissues of animals and plants into 0.5 cm thick rectangular pieces with a double-edged blade, and fix them in 2 mL of FAA fixative (70% anhydrous ethanol, formaldehyde and glacial acetic acid in a ratio of 18:1:1) overnight and evacuate the next day. Immediately fix the fresh tissues of animals with 4% paraformaldehyde fixative for more than 24 hours. S2 dehydration treatment: the fixed samples were dehydrated by gradient static dehydration in 85% ethanol, 95% ethanol and anhydrous ethanol twice; S3 transparent treatment: plant tissue samples were treated with a 1:1 dimethyl carbonate / anhydrous ethanol mixture for 2 hours, then treated with dimethyl carbonate for 2 hours, and then treated with dimethyl carbonate for another hour; animal tissue samples were treated with a 1:1 dimethyl carbonate / anhydrous ethanol mixture for 5-10 minutes, then treated with dimethyl carbonate for 5-10 minutes, and treated twice; S4 Wax immersion treatment: After the plant tissue samples were treated with dimethyl carbonate for transparency, the original solution was aspirated, an appropriate amount of crushed wax was added, and the samples were placed in a 60°C oven. The centrifuge tube cover was opened to evaporate overnight. The wax solution was replaced twice at an interval of 2 hours the next morning and then left overnight. After the animal tissue samples were treated with transparency, the paraffin was melted at 65°C for 1 hour and then treated three times. S5 Paraffin embedding and sectioning: The above steps can be embedded the next day after overnight. The sample is fixed in the embedding mold. After cooling and solidification, a complete wax block is obtained. The completely solidified paraffin sample is heated and fixed on the embedding tissue box. The slices are sliced ​​using a microtome with a thickness of 20µm. The cut wax strip is adhered to the glass slide by smoothing it with water. The water is dried on a slide dryer and the wax strip is fully unfolded, and then the slide is dried.

[0012] S6 Dewaxing, rehydration, staining, transparentization, and sealing: Soak the slides with wax tape in xylene, xylene, xylene / anhydrous ethanol mixture (1:1), anhydrous ethanol, 95% anhydrous ethanol, 90% anhydrous ethanol, 70% anhydrous ethanol, ultrapure water, ultrapure water, 0.5% toluidine blue solution, ultrapure water, ultrapure water, 70% anhydrous ethanol, 90% anhydrous ethanol, 95% anhydrous ethanol, anhydrous ethanol, xylene / anhydrous ethanol mixture (1:1), xylene, and xylene for five minutes in sequence. After the solution on the sample slide evaporates completely, add two drops of Canada resin and cover the slide to complete the sealing.

[0013] Preferably, the first three dehydration treatments in step S2 are performed for 2 hours each, and the last dehydration treatment is performed for 1 hour.

[0014] Preferably, in the paraffin embedding and sectioning process in step S5, the temperature of the slide drying machine is 40° C., and then the slides are placed in a 36° C. oven for drying for 3 days.

[0015] Compared with the prior art, the present invention has the following advantages: 1. The present invention greatly simplifies the S4 wax dipping treatment step.

[0016] 2. The dimethyl carbonate used in the present invention has the characteristics of no irritating odor, no toxicity, no carcinogenicity, miscibility with other organic reagents, and biodegradability. Toxicological experimental analysis shows that dimethyl carbonate has no significant effect on the key organs of mice, does not induce obvious inflammatory reactions, and exhibits higher biosafety.

[0017] 3. The animal and plant tissue slice wax tape obtained in the present invention is smoother, has good continuity and is not easy to break.

[0018] 4. The animal and plant tissue sections obtained in the present invention are more evenly stained, can maintain the integrity of the animal tissue structure, and better maintain the morphology and integrity of plant parenchyma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 AC is the volatility of xylene and dimethyl carbonate at 60°C and the change curve within 1.5 hours. DG is the wax band state of the leaf main vein (plant tissue) section and the wax band state of the heart section (animal tissue) treated with xylene and dimethyl carbonate. Among them, A is the volatility of xylene at 60°C, B is the volatility of dimethyl carbonate at 60°C, and C is the change curve of xylene and dimethyl carbonate within 1.5 hours; DE is the leaf main vein, and FG is the heart section.

[0020] Figure 2Comparison of paraffin sections of plant growth point tissues prepared with xylene and dimethyl carbonate, where (AC) are sections of growth point tissues of Arabidopsis thaliana, tobacco and rice prepared with xylene, (DF) are sections of growth point tissues of Arabidopsis thaliana, tobacco and rice prepared with dimethyl carbonate, bar = 200 µm, (GI) are the success rates of growth point sections of Arabidopsis thaliana, tobacco and rice, sample size n = 5 (growth point tissue) × 3 (continuous sections), (JL) are the perimeter-to-area ratios of parenchymal cells in growth point sections of Arabidopsis thaliana, tobacco and rice, P, perimeter; A, area; sample size n = 3 (growth point tissue) × 10 (thin wall cells); Figure 3 Comparison of paraffin sections of leaf midrib prepared with xylene and dimethyl carbonate, (AC) Sections of Arabidopsis, tobacco and rice leaf midrib prepared with xylene, (DF) Sections of Arabidopsis, tobacco and rice leaf midrib prepared with dimethyl carbonate, bar = 200 µm; (GI) Success rate of Arabidopsis, tobacco and rice leaf midrib section, sample size n = 5 (midrib tissue) × 3 (continuous sections), (JL) Perimeter-to-area ratio of parenchymal cells in Arabidopsis, tobacco and rice leaf midrib sections, P, perimeter; A, area, '*' and '***' indicate statistically significant differences at p < 0.05 and p < 0.001, sample size n = 3 (midrib tissue) × 10 (thin wall cells); Figure 4 Comparison of mouse tissue paraffin sections prepared with xylene and dimethyl carbonate. A is a mouse kidney section prepared with xylene and dimethyl carbonate, C is a mouse liver section prepared with xylene, and E is a mouse colon section prepared with xylene and dimethyl carbonate. The section thickness is 20 μm, and it is stained with HE staining group, bar = 200 μm, B is the success rate of mouse kidney section (SRS), D is the success rate of mouse liver section (SRS), F is the success rate of mouse colon section (SRS), and the number of samples n = 5 (tissue) × 3 (continuous sections); Figure 5 Analysis of body weight and organ index of mice treated with different preparations, where A is the body weight change of mice in the three groups within 28 days, data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, B is the comparison of organ index of each group, organ index is calculated as organ weight / body weight × 100%, number of samples in each group n = 10, *p < 0.05; Figure 6are the concentrations of inflammatory indicators in mice treated with different preparations, where A is the level of TNF-α in mouse serum, B is the concentration of IL-1β in mouse serum, C is the concentration of IL-6 in mouse serum, *p < 0.05, and D is the level of CRP in mouse serum. The data are expressed as mean ± standard deviation, and the number of samples in each group is n = 10; Figure 7 is a histological comparison of tissue sections of different organs of mice prepared using two transparent agents, where (AF) are sections of NC (control group), xylene group and dimethyl carbonate group, respectively, where A is spleen, B is liver, C is lung, D is kidney, E is colon, and F is heart. All tissue sections were stained with hematoxylin and eosin (H&E), and the scale bars from left to right are 1000μm, 200μm and 100μm, respectively. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0022] Example 1 A method for preparing paraffin sections of animal and plant tissues using dimethyl carbonate as a transparent reagent comprises the following steps: (1) Sample fixation: Cut the fresh plant tissue into 0.5 cm thick rectangular pieces with a double-edged blade, and fix them in 2 mL of FAA fixative (70% anhydrous ethanol, formaldehyde and glacial acetic acid in a ratio of 18:1:1) overnight and evacuate the next day. Immediately fix the fresh animal tissue with 4% paraformaldehyde fixative for more than 24 hours.

[0023] (2) Dehydration: The fixed samples were dehydrated in gradient standing with 85% ethanol, 95% ethanol and anhydrous ethanol once, with the first three dehydrations lasting 2 h each and the last dehydration lasting 1 h.

[0024] (3) Transparent treatment: Plant tissue samples were treated with a xylene / anhydrous ethanol mixture (mixed ratio 1:1) and a dimethyl carbonate / anhydrous ethanol mixture (mixed ratio 1:1) for 2 hours, followed by a xylene and dimethyl carbonate treatment for 2 hours, and then a xylene and dimethyl carbonate treatment for another hour. Animal tissue samples were treated with a xylene / anhydrous ethanol mixture (mixed ratio 1:1) and a dimethyl carbonate / anhydrous ethanol mixture (mixed ratio 1:1) for 5-10 minutes, followed by a xylene and dimethyl carbonate treatment for 5-10 minutes, for two treatments.

[0025] (4) Wax immersion treatment: I. After the plant tissue sample is treated with xylene for transparency, the original solution is aspirated and xylene solution and crushed wax are added. The xylene solution covers the crushed wax and is placed in a 38°C oven overnight. The next morning, the oven is changed to 60°C. The lid of the centrifuge tube is opened to evaporate the xylene for 2 hours. The wax solution is replaced and the xylene is continued to evaporate for 2 hours. The wax solution is replaced 3 times at intervals of 2.5 hours and then left overnight. II. After the plant tissue sample is treated with dimethyl carbonate for transparency, the original solution is aspirated and an appropriate amount of crushed wax is added. The sample is placed in a 60°C oven. The lid of the centrifuge tube is opened to evaporate the paraffin overnight. The wax solution is replaced twice at intervals of 2 hours the next morning and then left overnight. III. After the animal tissue sample is treated with transparency, the paraffin is melted at 65°C for 1 hour and then treated three times.

[0026] (5) Paraffin embedding and sectioning: After the above steps are completed overnight, embedding can be performed the next day. The sample is fixed in the embedding mold and a complete wax block is obtained after cooling and solidification. The completely solidified paraffin sample is heated and fixed on the embedding tissue box, and sliced ​​using a microtome with a thickness of 20 µm; the cut wax strip is adhered to the glass slide by leveling the water, and the water is dried on a 40°C drying machine and the wax strip is fully unfolded, and then the glass slide is placed in a 36°C oven for drying for 3 days.

[0027] (6) Dewaxing, rehydration, staining, transparentization, and sealing: Soak the slides with wax tape in xylene, xylene, xylene / anhydrous ethanol mixture (1:1), anhydrous ethanol, 95% anhydrous ethanol, 90% anhydrous ethanol, 70% anhydrous ethanol, ultrapure water, ultrapure water, 0.5% toluidine blue solution, ultrapure water, ultrapure water, 70% anhydrous ethanol, 90% anhydrous ethanol, 95% anhydrous ethanol, anhydrous ethanol, xylene / anhydrous ethanol mixture (1:1), xylene, and xylene for five minutes in sequence. After the solution on the sample slide evaporates completely, add two drops of Canada resin and cover with a slide to complete the sealing.

[0028] Example 2 In the paraffin section process, since the wax immersion needs to be carried out at 60°C, we compared the volatility of the two reagents at this temperature and their mixing state with the wax liquid. Figure 1 .

[0029] Conclusion: Dimethyl carbonate evaporates faster than xylene at 60°C. Interestingly, unlike xylene which is miscible with paraffin, dimethyl carbonate and paraffin liquid are stratified and located in the lower layer of paraffin ( Figure 1 A. Figure 1 B). In addition, plant tissues become colorless and transparent after being transparentized with xylene, which increases the difficulty of subsequent operations and requires the addition of dyes for auxiliary observation, increasing the risk of experimental failure ( Figure 1 D). On the contrary, the tissue treated with dimethyl carbonate is milky white and easy to observe with the naked eye, without the aid of dyes, which reduces the difficulty of operation and the risk of failure. Surprisingly, whether it is plant tissue or animal tissue, the sectioning effect of tissue treated with dimethyl carbonate is better than that of xylene, mainly reflected in the smoother wax strip, good continuity and less fragile. Therefore, based on the physical and chemical properties of dimethyl carbonate as a transparent agent and the state of the wax strip, it can be preliminarily confirmed that it has a strong advantage in replacing xylene.

[0030] Example 3 We used the growing points and main veins of leaves of Arabidopsis thaliana (Arabidopsis thaliana, Col 0), rice (Oryza sativa L.‌‌spp.japonica, var Nipponbare) and tobacco (Nicotiana tabacum L., var Honghuadajinyuan) as the objects. All materials were grown normally in a greenhouse with a 16h light / 8h dark photoperiod cycle.

[0031] Dimethyl carbonate as a clearing agent to improve the staining quality, cell boundaries and filling morphology of plant tissues Wax immersion is one of the important steps in paraffin sectioning. The degree of wax immersion directly affects the filling degree of tissue cells and the cell morphology and integrity after sectioning. In order to compare the effects of xylene and dimethyl carbonate as transparent agents in plant tissue sections, the results of microscopic observation of sections are shown in Figure 2 AF and Figure 3 AF.

[0032] Comparison of paraffin sections of plant tissues treated with two transparent agents showed that the observation results under low-power objective microscope of the two reagents were similar, but the growth point and main vein plant tissues treated with dimethyl carbonate were more uniformly stained ( Figure 2 AF and Figure 3 AF).

[0033] The thin-walled cells of the growing points were relatively regular and intact after treatment with the two transparent agents, without any cell squeezing or deformation. However, the thin-walled cells of the main veins of the leaves showed obvious differences after treatment with the two transparent agents. The cells treated with xylene showed deformation, especially the thin-walled cells of Arabidopsis and tobacco, while the cells treated with dimethyl carbonate maintained a good morphology and integrity ( Figure 2 AF and Figure 3 AF).

[0034] Conclusion: This indicates that dimethyl carbonate has potential advantages in improving staining quality, which provides a favorable basis for further optimizing the paraffin section staining process and improves the overall quality of tissue sections.

[0035] The success rate of sectioning is an important indicator for evaluating the effect of paraffin sectioning. This study conducted a statistical analysis on the success rate of sectioning of Arabidopsis, rice, and tobacco tissues. Figure 2 GI and Figure 3 GI.

[0036] Conclusion: Regardless of the growth point tissue or the main vein of the leaf, the success rate of slices of samples treated with dimethyl carbonate was slightly higher than that of samples treated with xylene, but there was no significant difference ( Figure 2 GI and Figure 3 GI). Therefore, dimethyl carbonate showed a good sectioning effect in plant tissues and had the potential to replace xylene.

[0037] Cell fullness: ImageJ was used to measure the circumference and corresponding area of ​​Arabidopsis and tobacco growing points and the central area of ​​the main vein of rice leaves, the thin-walled cell area outside the vascular tissue of the main vein of Arabidopsis and tobacco leaves, and the wide and narrow cells of rice growing points under a 40x field of view. The ratio of the circumference to the area of ​​the corresponding cells was used as the fullness of the cells impregnated with wax. Three samples were selected for each material, and 10 sets of data were measured for each sample. The bar graph results were drawn based on the statistical data.

[0038] In order to better demonstrate the above phenomenon, we used the cell perimeter area ratio to quantify cell morphology. The cell perimeter area ratio can reflect the changes in a cell's morphological structure. The smaller the ratio, the more compact the cell's morphological structure is and the less likely it is to deform. This study measured the perimeter area ratio of thin-walled cells in paraffin sections of the growth points and main veins of Arabidopsis, tobacco, and rice. It was found that there was no significant difference in the growth point cells of plants after treatment with the two transparent agents ( Figure 2 JL), but the main vein cells of plant leaves showed significant differences after treatment with the two transparent agents. The perimeter area ratio of Arabidopsis and tobacco cells treated with dimethyl carbonate was significantly smaller than that of cells treated with xylene, and that of rice was slightly smaller, but there was no significant difference ( Figure 3 JL).

[0039] Conclusion: Dimethyl carbonate and xylene have less effect on cell morphology and integrity in young tissues (growing points). However, in older tissues (main veins of leaves), dimethyl carbonate has a better effect. Therefore, dimethyl carbonate is better than xylene in maintaining cell morphology and structure as a transparent agent.

[0040] Example 4 Effect of dimethyl carbonate as a clearing agent in animal tissue sections We also replaced the reagents of dimethyl carbonate and xylene in the preparation process of animal tissue slices, and the test method was the same as in Example 1. The results are shown in Figure 4 .

[0041] in conclusion: Under the 4x field of view, the color of the slices treated with dimethyl carbonate is uniform, the normal tissue structure is clearly visible, and the cell outline morphology is normal, which is basically the same as the permeabilization effect of xylene. This effect is more significant in the slices with 100x and 200x magnification. The cytoplasm staining after dimethyl carbonate treatment is uniform, without too deep or too light phenomenon. These phenomena indicate that dimethyl carbonate has no obvious effect on the quality of animal tissue staining slices, and it is very feasible to replace xylene.

[0042] The integrity rate of sections is an important indicator for evaluating the effect of paraffin sections. This study conducted a statistical analysis on the success rate of sections of various organ tissues in mice (the statistical standard was the same as that of plant materials). The results showed that although the integrity rate of sections treated with xylene was slightly higher in some animal tissues, the integrity rate of sections treated with dimethyl carbonate was equivalent or higher in other tissues. For example, in the integrity rate of sections of mouse kidneys, dimethyl carbonate showed a better effect. Therefore, dimethyl carbonate also showed good sectioning effects in animal tissues and has the potential to replace xylene.

[0043] Example 5 Dimethyl carbonate has better biosafety To evaluate the toxicity of the two agents, we orally administered the drugs to each group of mice and observed their basic physiological status. The experiment was divided into a control group, a xylene group, and a dimethyl carbonate group.

[0044] The experiment used 6-week-old male KM mice, weighing 35-37g, and was kept in an SPF animal room with a temperature of about 24°C and a humidity of about 64%, while maintaining a 12-h light / dark cycle. The liver, kidney, and colon organ tissues of normal control mice were used for the paraffin section transparent reagent replacement experiment. All experimental procedures followed the ethical guidelines stipulated by the Animal Experiment Ethics Academic Committee of Tianjin University of Traditional Chinese Medicine. The study was approved by the Ethics Committee with the approval number: TCM-LAEC2024067z1019.

[0045] After one week of adaptive culture, 30 mice were randomly divided into a control group (NC, n=10), a xylene group (xylene, n=10), and a dimethyl carbonate group (dimethyl carbonate, n=10). All mice were given SPF-grade experimental animal maintenance feed and distilled water during the experiment, and their body weight was recorded daily. In addition, mice in the NC group were given 0.5% sodium carboxymethyl cellulose (10 mL / kg) by oral gavage daily, and mice in the xylene group and dimethyl carbonate group were given xylene (500 mg / kg) and dimethyl carbonate (500 mg / kg) daily, respectively, for 28 days. After the drug administration, the mice were anesthetized with 5% sodium pentobarbital, and the eyeballs were removed for blood collection. The blood was centrifuged at 4°C and 3500 rpm, and the supernatant was aspirated to obtain serum. After being killed by cervical dislocation, the heart, liver, spleen, lung, kidney, and colon of the mice were collected and stored in 4% PFA fixative. Body weight and organ indexes are shown in Figure 5 .

[0046] Results: After 2 weeks of xylene administration, the weight of mice began to drop below that of mice in the normal group, and this trend was maintained until the end of the experiment. However, the weight of mice given dimethyl carbonate was close to that of normal mice, with no significant difference ( Figure 5 A). After the experiment, we weighed and analyzed the heart, liver, spleen, lungs and kidneys of the mice. Compared with the control group ( Figure 5 B) The heart, spleen, lung and kidney indexes of mice did not change significantly; however, oral administration of xylene led to a significant increase in the liver index of mice, while the liver index of mice in the dimethyl carbonate group was close to normal levels. This suggests that xylene has a certain effect on the liver, while dimethyl carbonate has no significant effect on key organs.

[0047] ELASA assay of mouse serum samples Four inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and C-reactive protein (CRP), were detected using Elisa kits according to the instructions. The above experimental serum was removed and centrifuged to remove impurities. Subsequently, the 96-well plate was coated with a specific capture antibody, incubated and washed with buffer. The sample was added to allow the target antigen to bind to the capture antibody, and the unbound components were removed by incubation and washing. Next, an enzyme-labeled secondary antibody was added, and the plates were incubated and washed again. Then, an enzyme substrate was added for a color reaction, and a stop solution was added after the reaction. Finally, the absorbance value was read using an enzyme reader under the corresponding absorbance light, and the antigen concentration in the sample was calculated according to the standard curve. See Figure 6 .

[0048] The results showed that compared with the normal group, there was no significant change in CRP in mice treated with xylene ( Figure 6 D), but it will lead to an increase in the levels of TNF-α, IL-1β and IL-6 in mouse serum ( Figure 6A, B, C), while after dimethyl carbonate treatment, the levels of the four inflammatory indicators of mice were lower than those of the xylene group and closer to normal. Compared with xylene, dimethyl carbonate does not induce obvious inflammatory response in mice.

[0049] To further evaluate the effects of the two reagents on mouse organs, we performed hematoxylin-eosin-stained section analysis and invited pharmacology experts to perform pathological evaluation.

[0050] Tissue sections and He staining of various organs of mice (1) Fixation and sampling: Fresh tissue is immediately placed in a tissue fixative or a special fixative corresponding to the tissue for more than 24 hours. The tissue is removed from the fixative and trimmed flat with a scalpel in a fume hood. The trimmed tissue and the corresponding label are placed in the embedding frame.

[0051] (2) Dehydration and wax dipping: Place the dehydration box in the dehydrator and dehydrate with graded alcohols in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol for 30 minutes twice, alcohol benzene for 5-10 minutes, xylene for 5-10 minutes twice, and paraffin melted at 65℃ for 1 hour three times.

[0052] (3) Paraffin embedding: embed the wax-soaked tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and place it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0053] (4) Paraffin sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4 μm. Float the slices on 40°C warm water on the slicer to flatten the tissue, pick up the tissue with a glass slide, and bake the slices in a 60°C oven. After the wax is baked dry, take it out and store it at room temperature for later use.

[0054] (5) Dewaxing of paraffin sections: The sections were dewaxed in xylene twice, each time for 20 min; treated with anhydrous ethanol twice, each time for 5 min; treated with 75% alcohol for 5 min, and washed with tap water.

[0055] (6) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0056] (7) Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol for 5 min each, then stain them in eosin solution for 5 min.

[0057] (8) Dehydration and sealing: The sections were dehydrated in anhydrous ethanol three times for 5 min each time, cleared in xylene twice for 5 min each time, and sealed with neutral gum.

[0058] Data calculation and statistical analysis Success rate of slicing: 3 replicates were set for each tissue, 5 slides were set for each replicate, and 15 slides were set for each slide. Five groups of slides (3 replicates per slide, 45 slides in total) were used as calculation units, and the ratio of the number of successful slicing in each group of slides to the total number of slices in this group was the success rate of slicing in that group; the data of the success rate of slicing in 5 groups of each tissue were statistically analyzed and a bar graph was drawn, see Figure 4 B. Figure 4 D. Figure 4 F.

[0059] Data statistics and drawing: One-way ANOVA was used to compare the significant differences among the three groups of mice NC, xylene, and dimethyl carbonate. The bar graphs all use SEM± to represent the mean value, *p<0.05 is considered to be a significant statistical difference between the groups, and GraphPad Prism 9.5 was used to draw the graph. The significance of the T test (two-sample variance assumption) is indicated by *P<0.05, **P<0.01, and ***P<0.001.

[0060] The evaluation results showed that compared with normal mice, the density of B lymphocytes in the white pulp area of ​​the spleen of mice treated with xylene was relatively high, and the distribution of immune cells in the red pulp area increased, while the spleen of mice treated with dimethyl carbonate tended to be normal ( Figure 7 A). Interestingly, consistent with the liver index, we found that xylene caused obvious cavitation in mouse hepatocytes, and this change was more obvious in the portal vein area. In contrast, the liver tissue after dimethyl carbonate treatment was basically normal, and the hepatocyte cytoplasm was full without obvious degeneration ( Figure 7 B). Lung sections showed that xylene and dimethyl carbonate had little effect on the lungs. The alveoli of mice in each group were normal in size and no obvious physiological ruptures were observed. However, lymphocytes were occasionally seen around the blood vessels in the lungs of mice treated with xylene ( Figure 7 C). Kidney sections showed that the glomeruli treated with xylene were normal in size and no tissue fibrosis was observed, but lymphocytes were occasionally seen around the afferent and efferent arteries ( Figure 7 D). In addition, xylene and dimethyl carbonate had no significant effect on the colon and heart of mice; the morphology of colonic crypts was intact without changes, the goblet cells inside were filled without damage, and no obvious inflammatory cell infiltration was observed ( Figure 7 E); The nuclei of myocardial cells in each group of mice were normal and single, and the intercalated discs were connected intact ( Figure 7 F). The above pathological section results show that dimethyl carbonate has less impact on mouse organs and shows higher safety.

Claims

1. A new use of dimethyl carbonate, characterized in that: Used as a transparent reagent for preparing animal and plant tissue sections.

2. The new use according to claim 1, characterized in that: It is used as a clearing agent to prepare paraffin sections of animal and plant tissues.

3. A method for preparing paraffin sections of animal and plant tissues using dimethyl carbonate as a transparent reagent, characterized in that: The steps include: S1 Sample fixation: Cut the fresh tissues of animals and plants into 0.5 cm thick rectangular pieces with a double-edged blade, fix them in 2 mL of FAA fixative overnight and evacuate the next day. Immediately fix the fresh tissues of animals with 4% paraformaldehyde fixative for more than 24 hours. S2 dehydration treatment: the fixed samples were dehydrated by gradient static dehydration in 85% ethanol, 95% ethanol and anhydrous ethanol twice; S3 transparent treatment: plant tissue samples were treated with a 1:1 dimethyl carbonate / anhydrous ethanol mixture for 2 hours, then treated with dimethyl carbonate for 2 hours, and then treated with dimethyl carbonate for another hour; animal tissue samples were treated with a 1:1 dimethyl carbonate / anhydrous ethanol mixture for 5-10 minutes, then treated with dimethyl carbonate for 5-10 minutes, and treated twice; S4 Wax immersion treatment: After the plant tissue samples were treated with dimethyl carbonate for transparency, the original solution was aspirated, an appropriate amount of crushed wax was added, and the samples were placed in a 60°C oven. The centrifuge tube cover was opened to evaporate overnight. The wax solution was replaced twice at an interval of 2 hours the next morning and then left overnight. After the animal tissue samples were treated with transparency, the paraffin was melted at 65°C for 1 hour and then treated three times. S5 Paraffin embedding and sectioning: After the above steps are done overnight, embedding can be carried out the next day. The sample is fixed in the embedding mold. After cooling and solidification, a complete wax block is obtained. The completely solidified paraffin sample is heated and pasted to the embedding tissue box, and sliced ​​using a microtome with a thickness of 20µm. The cut wax strip is adhered to the glass slide by moisture leveling, and the moisture is dried on a drying machine and the wax strip is fully unfolded, and then the glass slide is dried; S6 Dewaxing, rehydration, staining, transparentization, and sealing: Soak the slides with wax tape in xylene, xylene, xylene / anhydrous ethanol mixture (1:1), anhydrous ethanol, 95% anhydrous ethanol, 90% anhydrous ethanol, 70% anhydrous ethanol, ultrapure water, ultrapure water, 0.5% toluidine blue solution, ultrapure water, ultrapure water, 70% anhydrous ethanol, 90% anhydrous ethanol, 95% anhydrous ethanol, anhydrous ethanol, xylene / anhydrous ethanol mixture (1:1), xylene, and xylene for five minutes in sequence. After the solution on the sample slide evaporates completely, add two drops of Canada resin and cover the slide to complete the sealing.

4. The method for paraffin sectioning of animal and plant tissues according to claim 3, characterized in that: The first three dehydration treatments in step S2 are performed for 2 hours each, and the last dehydration treatment is performed for 1 hour.

5. The method for paraffin sectioning of animal and plant tissues according to claim 3, characterized in that: In the paraffin embedding and sectioning process of step S5, the temperature of the slide drying machine is 40° C., and then the slides are placed in a 36° C. oven for drying for 3 days.

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