Preparation method for gonad sampling and tissue slices of Chinese softshell turtles in embryonic period
Through the alcohol sol mounting method and the protein-paraffin dual embedding method, the cell deformation and tissue loss of the gonads in the embedding section of the Chinese turtle during embryonic period were solved, and high-quality tissue section preparation was achieved, reducing costs and improving observation efficiency.
Patent Information
- Application Number
- CN202510248865.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-04
- Publication Date
- 2025-08-01
AI Technical Summary
In the prior art, during the embryonic section of Chinese turtle embryos, cells are prone to deformity and tissues are prone to fragmentation and loss. The traditional paraffin embedding method leads to poor section quality, affecting the observation of histological characteristics and pathological changes.
The embryonic gonadal tissue of Chinese turtle was fixed by alcohol sol mounting method, combined with the protein-paraffin double embedding method, pruning it by fixing alcohol sol-like substances, forming a protective shell with protein solution, and dehydration and transparent treatment with gradient ethanol and xylene, and finally hematoxylin staining and neutral resin sealing were used.
The non-destructive sampling and positioning of the embryonic gonads of Chinese turtle is achieved, the delicate tissue cells are protected, the cost of embedding is reduced, and the integrity of the section and the convenience of observation are improved.
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Figure CN120404218A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microscopic tissue sectioning, and specifically, to a method for sampling the gonads of soft-shelled turtle embryos and preparing tissue sections. Background Art
[0002] At present, for the section embedding method of the gonads of soft-shelled turtle embryos, most still adopt the traditional paraffin embedding method. However, since the gonadal cells of soft-shelled turtle embryos are more delicate than other tissues, and their osmotic pressure and chemical substances of the fixative during the paraffin embedding fixation process are also more sensitive than other tissues. Therefore, when using the traditional paraffin embedding method to prepare sections of the gonads of soft-shelled turtle embryos, problems such as cell deformation due to improper fixation are likely to occur; in the dehydration step of traditional paraffin embedding, the traditional ethanol gradient and dehydration time may cause over-dehydration or insufficient dehydration of the delicate gonadal cells of soft-shelled turtle embryos. Over-dehydration will cause the gonadal tissue to become brittle and affect the quality of the sections, while insufficient dehydration will hinder subsequent clearing and dewaxing, resulting in the inability of paraffin to fully infiltrate the gonads, and then tissue fragmentation or incomplete sections during sectioning, making the final obtained embedding blocks have limitations in observing and studying the histological characteristics, pathological changes, etc. of the gonads of soft-shelled turtle embryos. In addition, due to the extremely small volume of the gonadal tissue of soft-shelled turtle embryos and its connection with the body for life, when using traditional paraffin embedding, the situation of gonad loss is very likely to occur.
[0003] Based on this, it is necessary to develop a tissue embedding and sectioning method that is particularly suitable for the gonads of soft-shelled turtle embryos. Summary of the Invention
[0004] The technical problem to be solved by the present invention is how to overcome the problems of easy cell deformation and easy tissue fragmentation and loss during the embedding and sectioning process of the gonads of soft-shelled turtle embryos in the prior art.
[0005] To solve the above technical problems, the first aspect of the present invention provides a method for sampling the gonads of soft-shelled turtle embryos and preparing tissue sections, which specifically includes the following steps: S1. Sample exposure: Place the soft-shelled turtle with its abdomen facing upwards to fully expose the ventral carapace. Subsequently, use a scalpel to carefully operate along the outside of the xiphoid ventral carapace of the soft-shelled turtle to separate the ventral carapace. Then, switch to surgical scissors and perform a shearing action at the middle position of the xiphoid ventral carapace to separate the upper ventral carapace, middle ventral carapace, inner ventral carapace, and lower ventral carapace from each other, so as to completely expose the tissue. Since the gonadal tissue adheres to the sixth rib on the ventral surface of the dorsal carapace, subsequent related treatment steps are required.
[0006] S2. Sample solidification: Mix the alcohol sol-like substance with the soft-shelled turtle tissue sample, and after the alcohol sol-like substance solidifies, the solidification is completed; S3. Sample trimming: Trim the solidified Chinese soft-shelled turtle tissue sample in step S1. Specifically, trim the position where the top of the gonad in the mesonephros-gonad tissue is flush with the mesonephros, so that the gonad is fully exposed in the mesonephros part, forming a standard and unified shape. After pre-staining the trimmed mesonephros-gonad, rinse or soak it in 30% - 50% ethanol for 5 - 30 min, and then fix it with 4% paraformaldehyde. S4. Pre-embedding: Wrap the sample obtained in step S2 with a protein solution and trim the wrapped sample into a unified shape to obtain a pre-embedded block. S5. Dehydration and infiltration with wax: Dehydrate the pre-embedded block obtained in step S3 with ethanol of gradient concentrations. After dehydration, use xylene to make the pre-embedded block transparent, and after transparency, infiltrate the pre-embedded block with wax. S6. Shaping and sectioning: Place the pre-embedded block after wax infiltration in a mold for shaping and sectioning to obtain pre-sections. S7. Dewaxing: Dewax the pre-sections with xylene and ethanol. S8. Staining: Stain the dewaxed pre-sections with hematoxylin. S9. Mounting: Dehydrate the pre-sections after hematoxylin staining with ethanol. After dehydration, use xylene to make the pre-sections transparent. After transparency, perform mounting treatment on the pre-sections with neutral resin to obtain sections of the gonad of Chinese soft-shelled turtle embryos.
[0007] In view of the characteristics that the gonad tissue cells of Chinese soft-shelled turtle embryos are delicate and the tissue is extremely small, in step S1 of the present invention, the "mounting method" is adopted to achieve non-destructive indirect sampling. Subsequently, a protein mixture similar to the cell tissue morphology is used to embed the sample, improving the embedding method for special tissues such as the gonad of Chinese soft-shelled turtle embryos. Different from the conventional paraffin embedding, the present invention adopts a brand-new protein-paraffin double embedding method. This method can effectively protect the tiny tissue of the gonad of Chinese soft-shelled turtle embryos, realizing non-destructive sampling and positioning of the gonad of Chinese soft-shelled turtle embryos, effectively saving the embedding cost, and facilitating subsequent observation during sectioning.
[0008] Preferably, in step S1, the method for exposing the sample is a special anatomical method based on the body structure of Chinese soft-shelled turtle.
[0009] Preferably, in step S2, the alcohol sol-like substance is an alcohol-soluble mixture including any one or more of ethyl α-cyanoacrylate adhesive, SBS pressure-sensitive adhesive, and povidone.
[0010] Preferably, in step S3, the dye used for pre-staining is hematoxylin or eosin, and the staining duration for pre-staining is 30 - 150 s.
[0011] After the above-mentioned alcohol sol-like substance is mixed with the sex tissue sample of the Chinese soft-shelled turtle embryo, the alcohol sol-like substance will solidify, thereby realizing the fixation of the Chinese soft-shelled turtle tissue sample. The fixed Chinese soft-shelled turtle tissue sample can be shaped using conventional instruments such as surgical scissors and scalpels. After trimming and shaping, the alcohol sol-like substance can be completely removed with ethanol without residue. This "mounting method" indirect sampling can achieve non-destructive sampling of the gonads of tiny tissues in the embryonic stage.
[0012] Preferably, in the step S4, the protein solution includes any one of egg white, pea protein, and collagen.
[0013] Different from the direct paraffin embedding or agar-paraffin double embedding in the prior art, the present invention provides a brand-new protein-paraffin double embedding method. This method utilizes the structural characteristics of proteins such as egg white solution, collagen, and pea protein, and can form a gel on the surface of the gonad tissue through protein denaturation. After the gel is formed, it can be trimmed into a standard shape by trimming and shaping, which is convenient to operate. The gel can also effectively protect the gonad tissue, avoiding the problems of sample loss and tissue damage.
[0014] Preferably, the step S4 includes the following steps: immersing the sample obtained in step S3 in the protein solution, and making the protein denature and solidify by heating to form a protective shell on the surface of the sample, and obtaining a pre-embedded block after trimming.
[0015] Preferably, the step S5 includes the following steps: S51: Immerse the pre-embedded block in ethanol with concentrations of 50%, 70%, 80%, and 90% in sequence, and each immersion time is 15 - 20 min; S52: Immerse the pre-embedded block in 100% ethanol twice, and each immersion time is 5 - 10 min; S53: Immerse the pre-embedded block in a 50% (v / v) xylene-ethanol solution for 15 - 20 min; S54: Immerse the pre-embedded block in xylene twice, and each immersion time is 5 - 10 min; S55: Immerse the pre-embedded block in a 50% (v / v) paraffin-xylene solution at 70 °C for 20 - 40 min; S56: Immerse the pre-embedded block in paraffin at 65 °C twice, the first immersion time is 50 - 60 min, and the second immersion time is 4.5 - 5.5 h.
[0016] Preferably, the step S7 includes the following steps: S71: Immerse the pre-section in xylene twice, and each immersion time is 5 - 10 min; S72: Immerse the pre-section in a 50% (v / v) xylene-ethanol solution for 3 - 5 min; S73: Immerse the pre-section in 100% ethanol twice, with each immersion time being 3 - 5 min; S74: Immerse the pre-section in 90% and 80% ethanol successively, each for 1 - 3 min; S75: Immerse the pre-section in 70% and 50% ethanol successively, each for 1 - 2 min; S76: Immerse the pre-section in ddH2O for 1 - 3 min to complete dewaxing.
[0017] Aiming at the problems of over-dehydration or insufficient dehydration that may occur during the dehydration process in the paraffin embedding method in the prior art, the present invention provides a new dehydration and clearing procedure. Using this dehydration and clearing procedure, it can ensure that cell tissues are not damaged and the integrity of cell tissues is guaranteed.
[0018] Preferably, the step S9 includes the following steps: S91: Immerse the pre-section in 50%, 70%, 80%, and 90% ethanol successively, each for 15 - 20 min; S92: Immerse the pre-section in 100% ethanol twice, each for 5 - 10 min; S93: Immerse the pre-section in a 50% (v / v) xylene-ethanol solution for 15 - 20 min; S94: Immerse the pre-section in xylene twice, each for 5 - 10 min; S95: Mount the pre-section with neutral resin to obtain the gonad section of the Chinese soft-shelled turtle embryo.
[0019] Preferably, in the step S6, the thickness of the section is 3 - 6 μm, and the section also needs to be flattened and baked to form a pre-section.
[0020] Preferably, the flattening is carried out by using water at 42 - 46°C, and the baking step is: after flattening, take out the section and place it on a glass slide, and place the glass slide with the section in an environment of 60°C for drying for 8 - 12 min to obtain a pre-section.
[0021] Preferably, in the step S6, the mold includes a base and a sample cup. The shape of the base fits the embedding machine fixture. The sample cup is a cylindrical shape with a diameter of 0.3 - 1 cm and a height of 0.5 - 1.5 cm. The sample cup and the base are connected by threads, and a pull ring for demolding is provided on the side of the sample cup.
[0022] Due to the relatively shallow traditional paraffin embedding frames, the gonad samples of Chinese soft-shelled turtle embryos being small and slender, traditional paraffin embedding frames are not easily able to effectively embed samples that have been embedded with protein protection. To address related issues, the present invention provides a new type of mold, which, while effectively embedding samples, also facilitates the demolding operation after paraffin embedding.
[0023] The second aspect of the present invention provides a section of the gonad of a Chinese soft-shelled turtle embryo, which is prepared by the method for sampling and preparing tissue sections of the gonad of a Chinese soft-shelled turtle embryo described in the first aspect.
[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. The method for preparing a section of the gonad of a Chinese soft-shelled turtle embryo provided by the present invention uses a "mounting" method to fix the mesonephros-gonad tissue, which is convenient for trimming, and can collect the gonad tissue of a Chinese soft-shelled turtle embryo without damage. At the same time, the alcohol sol used can be quickly removed by ethanol, leaving no residue and having a low cost; 2. The method for preparing a section of the gonad of a Chinese soft-shelled turtle embryo provided by the present invention is prepared by a protein-paraffin double embedding method, which can protect the delicate gonad of a Chinese soft-shelled turtle embryo before dehydration and clearing. At the same time, pre-embedding with protein can easily obtain uniform sections, and the operation is convenient; 3. The method for preparing a section of the gonad of a Chinese soft-shelled turtle embryo provided by the present invention uses hematoxylin staining instead of HE staining, which is convenient for subsequent observation; 4. The present invention provides a new type of mold, which can be used to effectively embed sections of the gonad of a Chinese soft-shelled turtle embryo, can greatly reduce the consumption of embedding raw materials such as paraffin and protein, and significantly reduces the preparation cost of sections of the gonad of a Chinese soft-shelled turtle embryo. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 It is a comparison diagram of the fixation effect of step S1 in the specific embodiment of the present invention; Figure 2 It is a trimming effect diagram of step S2 in the specific embodiment of the present invention; Figure 3 It is a comparison diagram of the morphology of HE-stained sections prepared by the traditional paraffin embedding method and the morphology of hematoxylin-stained sections prepared by the protein-paraffin double embedding method in the specific embodiment of the present invention; [[ID=,28]] Figure 4 It is a photo of embryo gonad sections of different embryonic stages prepared by embedding samples with ethyl cyanoacrylate adhesive in Example 1 of the present invention; Figure 5 It is a photo of embryo gonad sections of different embryonic stages prepared by embedding samples with SBS pressure-sensitive adhesive in Example 2 of the present invention; Figure 6Photographs of embryonic gonad sections of different embryonic stages prepared by embedding samples with povidone in Example 3 of the present invention. Detailed implementation manners
[0026] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of specific embodiments of the present invention is provided. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the protection scope of the claims of the present invention.
[0027] It should be noted that the endpoints and any values within the ranges disclosed in this article are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.
[0028] Unless otherwise defined, all terms, symbols and other scientific terms used in this article are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventional understood meanings are defined in this article for the purpose of clarification or easy reference. Such definitions in this article should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or cited in this article are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out according to the protocols and parameters given by the manufacturers.
[0029] The general paraffin embedding technique is mainly designed and optimized for common animal tissues to observe histological characteristics and pathological changes. Currently, the preparation of gonad sections of Chinese soft-shelled turtle embryos mostly uses the traditional paraffin embedding method. However, this method causes too much damage to the gonad tissues of Chinese soft-shelled turtle embryos. The samples are easily broken during dehydration and clearing, and the gonad tissues are easily lost during trimming. These defects lead to a reduction in the efficiency of subsequent observation of histological characteristics and pathological changes. Currently, there are case reports on the use of agar-paraffin double embedding method for treating zebrafish larvae. However, the agar-paraffin double embedding method takes a long time, and at the same time, the embedding effect of the agar-paraffin double embedding method on the gonads of Chinese soft-shelled turtle embryos is also poor.
[0030] In view of this, the specific implementation manner of the present invention provides a method for sampling and preparing tissue sections of the gonads of Chinese soft-shelled turtle embryos, which specifically includes the following steps: S1. Sample exposure: Place the soft-shelled turtle on its back to fully expose the ventral carapace. Subsequently, use a scalpel to carefully operate along the outside of the xiphisternum of the soft-shelled turtle to separate the ventral carapace. Then, switch to surgical scissors and perform a cutting action in the middle of the xiphisternum to separate the upper ventral plate, middle ventral plate, inner ventral plate, and lower ventral plate from each other, thereby fully exposing the tissue. Since the gonadal tissue adheres to the sixth rib on the ventral surface of the dorsal carapace, subsequent relevant processing steps are required.
[0031] S2. Sample solidification: Mix the alcohol sol-like substance with the soft-shelled turtle tissue sample. After the alcohol sol-like substance solidifies, the solidification is completed. S3. Sample trimming: Trim the solidified soft-shelled turtle tissue sample in step S1. Specifically: Trim the position where the top of the gonad in the mesonephros-gonad tissue is flush with the mesonephros to fully expose the gonad in the mesonephros part and form a standard and uniform shape. After pre-staining the trimmed mesonephros-gonad, rinse or soak it in 30% - 50% ethanol for 5 - 30 min, and then fix it with 4% paraformaldehyde. S4. Pre-embedding: Wrap the sample obtained in step S2 with a protein solution and trim the wrapped sample to a uniform shape to obtain a pre-embedded block. S5. Dehydration and infiltration with wax: Dehydrate the pre-embedded block obtained in step S3 with ethanol of gradient concentrations. After dehydration, use xylene to make the pre-embedded block transparent, and after transparency, infiltrate the pre-embedded block with wax. S6. Shaping and sectioning: Place the pre-embedded block after wax infiltration in a mold for shaping and sectioning to obtain pre-sections. S7. Dewaxing: Use xylene and ethanol to dewax the pre-sections. S8. Staining: Perform hematoxylin staining on the dewaxed pre-sections. S9. Mounting: Dehydrate the pre-sections after hematoxylin staining with ethanol. After dehydration, use xylene to make the pre-sections transparent, and after transparency, perform a mounting treatment with neutral resin to obtain soft-shelled turtle embryonic gonad sections.
[0032] In step S1, the method of exposing the sample is a special anatomical method based on the body structure of the soft-shelled turtle.
[0033] In step S2 of the above embodiment, the alcohol sol-like substance is an alcohol-soluble mixture of any one or more of ethyl α-cyanoacrylate adhesive, SBS pressure-sensitive adhesive, and povidone.
[0034] More specifically, in the above embodiment, the alcohol sol-like substance is ethyl α-cyanoacrylate adhesive or SBS pressure-sensitive adhesive - rosin resin - white oil or povidone ethanol viscous glue.
[0035] The fixed effect comparison of step S2 is Figure 1 As shown, Figure 1 A in the figure is a photo in which alcohol-soluble colloidal substances are not mixed with Chinese soft-shell turtle tissue samples. Figure 1 B is a photograph of a tissue sample of a Chinese soft-shell turtle mixed with an alcohol-soluble colloidal substance. It can be seen from the photograph that the tissue sample fixed with the alcohol-soluble colloidal substance is easier to trim in step S3.
[0036] The trimming effect of step S3 is as follows Figure 2 As shown, Figure 2 A in the figure is the mesonephros-gonadal tissue before modification. Figure 2 B in the figure is the modified mesonephros-gonadal tissue. It can be seen from the figure that the present embodiment uses alcohol-soluble colloidal substances to fix tissue samples to obtain embryonic gonadal tissue samples of Chinese soft-shelled turtle without damage, overcoming the problems of easy deformation of gonadal cells and easy loss of tissue in existing embedding technologies.
[0037] Since the gonadal tissue of the Chinese soft-shelled turtle embryo is small and fragile, and forms a complex with the mesonephros and adrenal glands, the following problems arise during sampling: (1) The gonad is small and easily broken, so direct sampling causes significant damage to the sample; (2) The gonad is translucent, making it difficult to observe and easily lost during sample handling; (3) The gonad is small and the mesonephros is large, so when the gonad, mesonephros, and adrenal glands are processed and sliced together, the gonad is not easily cut; (4) A portion of the gonad is free from the mesonephros, and this portion is easily lost during the operation. Through steps S1 and S2 of the above embodiment, an undamaged, morphologically standardized, gonadal tissue sample of the Chinese soft-shelled turtle embryo can be obtained.
[0038] In step S4 of the above embodiment, the protein solution used is preferably any one of an egg white-rosin mixed solution, a collagen solution, and a pea protein solution.
[0039] In step S4 of the above embodiment, the sample is pre-embedded by utilizing the heat-coagulation properties of protein, forming a protective shell around the sample, thereby protecting and pre-embedding the delicate gonads before dehydration and transparency. In step S4, pre-embedding can be performed in a mold to produce a uniform, uniform pre-embedded block. Using a protein solution to pre-embed the sample also avoids problems such as sample loss and damage.
[0040] In step S5 of the above embodiment, in order to solve the problem of insufficient or excessive dehydration in the prior art, the above embodiment develops the following dehydration, clearing and wax dipping process: S51: Immerse the pre-embedded block in 50%, 70%, 80%, and 90% ethanol, respectively, for 15-20 minutes each time. S52: Immerse the pre-embedded block in 100% ethanol twice, with each immersion time being 5 - 10 min; S53: Immerse the pre-embedded block in a 50% (v / v) xylene-ethanol solution for 15 - 20 min; S54: Immerse the pre-embedded block in xylene twice, with each immersion time being 5 - 10 min; S55: Immerse the pre-embedded block in a 50% (v / v) paraffin-xylene solution at 70 °C for 20 - 40 min; S56: Immerse the pre-embedded block in paraffin at 65 °C twice. The first immersion time is 50 - 60 min, and the second immersion time is 4.5 - 5.5 h.
[0041] As a preferred embodiment, in the preparation method provided in the above embodiment, during the pre-embedding in step S4 and the wax infiltration in step S5, the sample can be placed in a cylindrical mold to achieve the purpose of improving efficiency and saving costs. More specifically, step S5 of the specific embodiment of the present invention provides a mold product for embedding. The mold product includes a base and a sample cup. The shape of the base fits the embedding machine fixture. The sample cup is a cylindrical shape with a diameter of 0.3 - 1 cm and a height of 0.5 - 1.5 cm. The sample cup and the base are connected by a thread, and a pull ring for demolding is provided on the side of the sample cup.
[0042] When embedding gonadal tissue, since the gonadal tissue is small, it is not easy to uniformly standardize the embedding position of the gonadal tissue. If one paraffin block embeds one sample, due to the small gonadal-mesonephric tissue and large paraffin block, it will cause waste of paraffin and extremely low efficiency, and may lead to tissue displacement or inaccurate positioning during subsequent embedding. Once the tissue position changes, it is difficult to ensure that the tissue layer cut during sectioning is an accurate and representative cross-section. At the same time, when performing multiple embeddings, the positions of the gonads may not be in the same plane, resulting in the inability to perform multi-sample embedding, thus affecting the accuracy of the experimental results. To address the above problems, the specific embodiment of the present invention provides a deep embedding method. Since a single gonadal tissue is difficult to fix its position during embedding, but the pre-embedded protein block is a three-dimensional and uniform structure, it can be conveniently positioned and embedded. This embodiment designs a device for deep embedding of small samples. After the wax infiltration of a single pre-embedded material is completed, a cylindrical card slot loading device that conforms to the size of the pre-embedded block and is uniform is designed and fixed on the microtome for sectioning. At the same time, when embedding multiple samples, the wax-infiltrated samples are arranged in sequence in the loading device and then tissue sectioning is performed according to the conventional operation to achieve the purpose of improving efficiency and saving costs.
[0043] In step S7 of the above embodiment, the dewaxing specifically includes the following steps: S71: Immerse the pre-section in xylene twice, with each immersion time being 5 - 10 min; S72: Immerse the pre-section in a 50% (v / v) xylene - ethanol solution for 3 - 5 min; S73: Immerse the pre-section in 100% ethanol twice, with each immersion time being 3 - 5 min; S74: Immerse the pre-section in 90% and 80% ethanol successively, once for each concentration, with each immersion time being 1 - 3 min; S75: Immerse the pre-section in 70% and 50% ethanol successively, once for each concentration, with each immersion time being 1 - 2 min; S76: Immerse the pre-section in ddH2O for 1 - 3 min, and the dewaxing is completed.
[0044] In step S8 of the above - mentioned embodiment, hematoxylin staining is used instead of HE staining. The reason is that eosin dye used in HE staining will completely stain the gonads red, which is not convenient for subsequent observation. Therefore, only hematoxylin staining is adopted.
[0045] Figure 3 For the morphology of HE - stained sections prepared by the traditional paraffin - embedding method and the morphology of hematoxylin - stained sections prepared by the protein - paraffin double - embedding method of this embodiment, where, Figure 4 A in is the morphology of HE - stained sections prepared by the traditional paraffin - embedding method, Figure 4 B in is the morphology of hematoxylin - stained sections prepared by the protein - paraffin double - embedding method of this embodiment. It can be seen from Figure 4 that the morphology of the sections prepared by the method provided in this embodiment is more uniform, and the blue - purple tissue cell morphology can be clearly observed with the result of hematoxylin staining.
[0046] In step S8 of the above - mentioned embodiment, step S9 specifically includes the following steps: S91: Immerse the pre-section in 50%, 70%, 80%, and 90% ethanol successively, once for each concentration, with each immersion time being 15 - 20 min; S92: Immerse the pre-section in 100% ethanol twice, with each immersion time being 5 - 10 min; S93: Immerse the pre-section in a 50% (v / v) xylene - ethanol solution for 15 - 20 min; S94: Immerse the pre-section in xylene twice, with each immersion time being 5 - 10 min; S95: Mount the pre-section with neutral resin to obtain the gonad sections of Chinese soft - shelled turtle embryos.
[0047] The technical solution of the present invention is further illustrated by specific embodiments below.
[0048] Example 1 In this example, ethyl α-cyanoacrylate adhesive was used to fix the samples. The Chinese soft-shelled turtle was placed with its abdomen facing upwards to fully expose the abdominal carapace. Subsequently, a scalpel was used to carefully operate along the outside of the ventral keel carapace of the Chinese soft-shelled turtle to separate the abdominal carapace. Then, surgical scissors were used to perform a shearing action at the middle position of the ventral keel carapace to separate the upper ventral carapace, middle ventral carapace, inner ventral carapace, and lower ventral carapace from each other, so that the tissues were completely exposed. Since the gonadal tissue was adhered to the sixth rib on the ventral surface of the dorsal carapace. Ethyl α-cyanoacrylate adhesive was used to fix the sexually indifferent mesonephros-gonadal tissues of Chinese soft-shelled turtle embryos at 30-day embryos, 40-day embryos, and 50-day embryos respectively. After the ethyl α-cyanoacrylate solidified, forceps and a scalpel were used to trim the solidified samples, and the positions where the top of the gonad in the mesonephros-gonadal tissue was flush with the mesonephros were trimmed. After the trimming was completed, the samples were soaked in 50% ethanol for 10-20 min to remove the ethyl α-cyanoacrylate adhesive, and then fixed with 4% paraformaldehyde for 5 h.
[0049] The aforementioned samples and an egg white solution mixed with 1% mass fraction of rosin were added to a cylindrical mold. The samples should be immersed below the liquid level of the solution. After heating the mold at 100 °C for 8 min, the solution solidified around the samples to form a protective shell, and a cylindrical pre-embedding block was obtained. The pre-embedding block was trimmed to make its shape uniform.
[0050] The whole pre-embedding was dehydrated. It was successively soaked in 50% ethanol, 70% ethanol, 80% ethanol, and 90% ethanol for 16 min each, and then dehydrated with 100% ethanol Ⅰ and 100% ethanol Ⅱ for 10 min each time. The dehydrated pre-embedding block was buffered with 50% (v / v) xylene-absolute ethanol for 20 min, and made transparent with xylene Ⅰ and xylene Ⅱ for 10 min each time. The pre-embedding block after the transparency treatment was buffered with 50% (v / v) paraffin-xylene at 70 °C; after 30 min, it was infiltrated with paraffin Ⅰ at 65 °C for 60 min, and paraffin Ⅱ at 65 °C for 5 h to obtain an embedded paraffin block. The infiltration process was carried out in a cylindrical mold.
[0051] A microtome was used to slice the paraffin block, and the slice thickness was set to 5 μm. The slices were flattened with 45 °C water. After the flattening was completed, the slices were placed on glass slides and baked at 60 °C for 10 min to obtain pre-slices.
[0052] The glass slide was eluted in xylene I and xylene II for 10 minutes each time, then buffered in 50% (v / v) xylene-absolute ethanol for 5 minutes, and then eluted with 100% ethanol I and 100% ethanol II for 5 minutes each time, eluted in 90% ethanol and 80% ethanol for 3 minutes, eluted in 70% ethanol and 50% ethanol for 2 minutes, then soaked in water for 3 minutes and stained with hematoxylin for 2 minutes, and then the dye was removed with water.
[0053] The above-mentioned pre-sectioned slices were successively soaked in distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 50% (v / v) xylene-absolute ethanol, xylene I and xylene II for dehydration and clearing, with each soaking duration being 2 minutes. Then, the slices were sealed with neutral resin to obtain the gonad slices of the Chinese soft-shelled turtle embryo. The slices were observed under an optical microscope, and the experimental results are as Figure 4 shown, where Figure 4 A in Figure 4 is a photo of the gonad slice of the 30-day embryo, Figure 4 B in
[0054] Example 2 In this example, the SBS pressure-sensitive adhesive was used to fix the samples S1. Sample exposure: The Chinese soft-shelled turtle was placed with its abdomen facing up to fully expose the abdominal carapace. Subsequently, a scalpel was used to carefully operate along the outside of the ventral carapace of the Chinese soft-shelled turtle to separate the abdominal carapace. Then, a pair of surgical scissors was used to perform a shearing action at the middle position of the ventral carapace to separate the upper ventral carapace, middle ventral carapace, inner ventral carapace, and lower ventral carapace, so that the tissue was completely exposed. Since the gonad tissue was adhered to the sixth rib on the ventral surface of the dorsal carapace, subsequent related treatment steps were required. A mixture of 50 parts by mass of SBS pressure-sensitive adhesive, 40 parts by mass of rosin resin, and 10 parts by mass of white oil was mixed to obtain an SBS mixture. The SBS mixture was used to fix the mesonephros-gonad tissues of the Chinese soft-shelled turtle embryos at 30 days, 40 days, and 50 days. After the SBS mixture solidified, forceps and a scalpel were used to trim the solidified samples, and the positions where the top of the gonad and the mesonephros were flush in the mesonephros-gonad tissues were trimmed. After the trimming was completed, the samples were soaked in 50% ethanol for 10 - 20 minutes to remove the SBS mixture, and then fixed with 4% paraformaldehyde for 5 hours.
[0055] Add the aforementioned sample and a collagen solution with a pH of 6.5 to a cylindrical mold. The sample should be submerged below the liquid level of the solution. After heating the mold at 80 °C for 5 minutes, the solution solidifies around the sample to form a protective shell, obtaining a cylindrical pre-embedded block. Trim the pre-embedded block to make its shape uniform.
[0056] Dehydrate the whole pre-embedded block. Immerse it successively in 50% ethanol, 70% ethanol, 80% ethanol, and 90% ethanol for 16 minutes each, and then dehydrate it in 100% ethanol Ⅰ and 100% ethanol Ⅱ for 10 minutes each. Buffer the dehydrated pre-embedded block with 50% (v / v) xylene-absolute ethanol for 20 minutes, and then clear it in xylene Ⅰ and xylene Ⅱ for 10 minutes each. Buffer the cleared pre-embedded block with 50% (v / v) paraffin-xylene at 70 °C; after 30 minutes, impregnate it with paraffin Ⅰ at 65 °C for 60 minutes and paraffin Ⅱ at 65 °C for 5 hours to obtain an embedded paraffin block. The impregnation process is carried out in a cylindrical mold.
[0057] Use a microtome to slice the paraffin block. Set the slice thickness to 5 μm. Spread the slices with 45 °C water. After spreading, place the slices on a glass slide and bake them at 60 °C for 10 minutes to obtain pre-slices.
[0058] Elute the glass slide in xylene Ⅰ and xylene Ⅱ for 10 minutes each, then buffer it in 50% (v / v) xylene-absolute ethanol for 5 minutes, and then elute it with 100% ethanol Ⅰ and 100% ethanol Ⅱ for 5 minutes each, elute it in 90% ethanol and then 80% ethanol for 3 minutes, elute it in 70% ethanol and then 50% ethanol for 2 minutes, and then soak it in water for 3 minutes and perform hematoxylin staining for 2 minutes, and then remove the dye with water.
[0059] Successively immerse the above pre-slices in distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol Ⅰ, 100% ethanol Ⅱ, 50% (v / v) xylene-absolute ethanol, xylene Ⅰ, and xylene Ⅱ for dehydration and clearing. The soaking time for each time is 2 minutes. Then, seal the slices with neutral resin to obtain the gonad slices of the Chinese soft-shelled turtle embryo. Observe the slices under an optical microscope. The experimental results are as Figure 5 shown, where Figure 5 A in it is a photo of the gonad slice of a 30-day embryo, Figure 5 B in it is a photo of the gonad slice of a 40-day embryo, Figure 5 C in it is a photo of the gonad slice of a 50-day embryo. When observing, only need to find ovalbumin for positioning and then directly find the sample in ovalbumin. The gonad tissue exists in the protein and shows blue-violet color. At the same time, the gonad tissue cells are complete, and cross-sections can be observed, enabling histological observation and analysis of the gonad.
[0060] Example 3 In this example, povidone was used to fix the samples. S1. Sample exposure: The soft-shelled turtle was placed with its abdomen facing upwards to fully expose the ventral carapace. Subsequently, a scalpel was used to carefully operate along the outside of the xiphisternal carapace of the soft-shelled turtle to separate the ventral carapace. Then, surgical scissors were used to perform a shearing action at the middle position of the xiphisternal carapace to separate the upper ventral carapace, middle ventral carapace, inner ventral carapace, and lower ventral carapace from each other, so that the tissue was completely exposed. Since the gonadal tissue was adhered to the sixth rib on the ventral surface of the dorsal carapace, subsequent related processing steps were required. 3 parts by mass of ethanol was mixed with 97 parts by mass of povidone to obtain a PVP viscous glue. The embryonic sex mesonephros-gonad tissues of soft-shelled turtles at 30-day embryos, 40-day embryos, and 50-day embryos were fixed with the PVP viscous glue respectively. After the PVP viscous glue solidified, forceps and a scalpel were used to shape the solidified samples, and the positions where the top of the gonad in the mesonephros-gonad tissue was flush with the mesonephros were trimmed. After shaping, the samples were soaked in 50% ethanol for 10 - 20 min to remove the PVP viscous glue, and then fixed with 4% paraformaldehyde for 5 h.
[0061] The aforementioned samples and a pea protein solution with a pH of 5.0 were added to a cylindrical mold. The samples should be submerged below the liquid level of the solution. After heating the mold at 80 °C for 5 min, the solution solidified around the samples to form a protective shell, obtaining a cylindrical pre-embedded block. The pre-embedded block was trimmed to make its shape uniform.
[0062] The whole pre-embedded block was dehydrated by soaking it in 50% ethanol, 70% ethanol, 80% ethanol, and 90% ethanol for 16 min each in sequence, and then dehydrated in 100% ethanol I and 100% ethanol II for 10 min each time. The dehydrated pre-embedded block was buffered with 50% (v / v) xylene-anhydrous ethanol for 20 min, and made transparent with xylene I and xylene II for 10 min each time. The pre-embedded block after transparency treatment was buffered with 50% (v / v) paraffin-xylene at 70 °C; after 30 min, infiltrated with paraffin I at 65 °C for 60 min, and paraffin II at 65 °C for 5 h to obtain an embedded paraffin block. The infiltration process was carried out in a cylindrical mold.
[0063] The paraffin block was sliced with a microtome, and the slice thickness was set to 5 μm. The slices were flattened with 45 °C water. After flattening, the slices were placed on glass slides and baked at 60 °C for 10 min to obtain pre-slices.
[0064] The slides were eluted in xylene I and xylene II for 10 minutes each, then buffered in 50% (v / v) xylene-anhydrous ethanol for 5 minutes, then eluted with 100% ethanol I and 100% ethanol II for 5 minutes each, eluted in 90% ethanol and 80% ethanol for 3 minutes, and eluted in 70% ethanol and 50% ethanol for 2 minutes. After soaking in water for 3 minutes, hematoxylin staining was performed for 2 minutes and the dye was removed with water.
[0065] The above pre-sections were soaked in distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 50% (v / v) xylene-anhydrous ethanol, xylene I and xylene II in sequence for dehydration and transparency. Each soaking time was 2 minutes. Then, neutral resin was used to seal the sections to obtain the gonad sections of the Chinese soft-shelled turtle embryo. The sections were observed under an optical microscope. The experimental results are shown in Figure 2. Figure 6 As shown, Figure 6 A in the figure is a photo of the gonad section of a 30-day embryo. Figure 6 B is a photo of the gonad section of a 40-day embryo. Figure 6 Image C shows a section of the gonads from a 50-day embryo. The sample can be found directly within the egg white protein, simply by locating it. The gonadal tissue, present within the protein, appears bluish-purple. The gonadal cells are intact, allowing for cross-section observation and histological analysis of the gonads.
[0066] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.
Claims
1. A method for sampling the gonads of soft-shelled turtle embryos during the embryonic period and preparing tissue sections, characterized in that, The following steps are involved: S1. Sample exposure: Place the Chinese soft-shell turtle with its abdomen facing upward to fully expose the plastron. Use a scalpel to separate the plastron along the outer side of the xiphoid plate. Use surgical scissors to cut the middle of the xiphoid plate to separate the upper, middle, inner, and lower plastrons, thereby completely exposing the tissue. S2. Sample solidification: Mixing the alcohol-soluble colloidal substance with the Chinese soft-shell turtle tissue sample, and solidifying after the alcohol-soluble colloidal substance solidifies; S3. Sample trimming: The Chinese soft-shell turtle tissue sample solidified in step S1 is trimmed, specifically: the top of the gonad in the mesonephros-gonadal tissue is trimmed flush with the mesonephros to fully expose the gonad in the mesonephros and form a standard uniform shape. The trimmed mesonephros-gonadal tissue is pre-stained, rinsed or soaked in 30%-50% ethanol for 5-30 minutes, and then fixed with 4% paraformaldehyde. S4, pre-embedding: encapsulating the sample obtained in step S2 with a protein solution and trimming the encapsulated sample into a uniform shape to obtain a pre-embedded block; S5, dehydration and wax dipping: Dehydrating the pre-embedded block obtained in step S3 using gradient concentrations of ethanol, transparentizing the pre-embedded block using xylene after dehydration, and then dipping the pre-embedded block in wax after transparency; S6. Shaping and slicing: placing the pre-embedded block after wax immersion in a mold to shape and slice it to obtain pre-slices; S7, dewaxing: Dewax the pre-sections using xylene and ethanol; S8. Staining: Perform hematoxylin staining on the dewaxed pre-sections; S9. Sealing: Dehydrate the pre-sections stained with hematoxylin with ethanol, make them transparent with xylene, and seal them with neutral resin to obtain gonad sections of Chinese soft-shell turtle embryos.
2. The method for sampling the gonads of soft-shelled turtle embryos during the embryonic period and preparing tissue sections according to claim 1, characterized in that, In step S2, the alcohol-soluble colloidal substance is an alcohol-soluble mixture of any one or more of ethyl α-cyanoacrylate adhesive, SBS pressure-sensitive adhesive, and polyvidone.
3. The method for sampling the gonads of Chinese soft-shelled turtle embryos during the embryonic period and preparing tissue sections according to claim 1, wherein, In step S4, the protein solution includes any one of egg white, pea protein and collagen.
4. The method for sampling the gonads and preparing tissue sections during the embryonic stage of the Chinese soft-shelled turtle as claimed in claim 1, wherein, The step S4 comprises the following steps: immersing the sample obtained in the step S3 in a protein solution, denaturing and solidifying the protein by heating to form a protective shell on the surface of the sample, and trimming to obtain a pre-embedded block.
5. The method for sampling the gonads and preparing tissue sections of Chinese soft-shelled turtle embryos as described in claim 1, characterized in that, The step S5 comprises the following steps: S51: Immerse the pre-embedded block in 50%, 70%, 80%, and 90% ethanol, respectively, for 15-20 minutes each time. S52: Immerse the pre-embedded block in 100% ethanol twice, each immersion time is 5-10 minutes; S53: Immerse the pre-embedded block in 50% (v / v) xylene-ethanol solution for 15-20 minutes; S54: Immerse the pre-embedded block in xylene twice, each immersion time is 5-10 minutes; S55: Immerse the pre-embedded block in 50% (v / v) paraffin-xylene solution at 70°C for 20-40 minutes; S56: Immerse the pre-embedded block in paraffin at 65 °C twice. The immersion time for the first time is 50 - 60 min, and the immersion time for the second time is 4.5 - 5.5 h.
6. The method for sampling the gonads of Chinese soft-shelled turtle embryos during the embryonic period and preparing tissue sections according to claim 1, characterized in that, The step S7 includes the following steps: S71: Immerse the pre-section in xylene twice, with an immersion time of 5 - 10 min each time; S72: Immerse the pre-section in a 50% (v / v) xylene-ethanol solution for 3 - 5 min; S73: Immerse the pre-section in 100% ethanol twice, with an immersion time of 3 - 5 min each time; S74: Immerse the pre-section in 90% and 80% ethanol successively once each, with an immersion time of 1 - 3 min each time; S75: Immerse the pre-section in 70% and 50% ethanol successively once each, with an immersion time of 1 - 2 min each time; S76: Immerse the pre-section in ddH2O for 1 - 3 min to complete dewaxing.
7. The method for sampling the gonads and preparing tissue sections during the embryonic stage of Chinese soft-shelled turtle as claimed in claim 1, wherein The step S9 includes the following steps: S91: Immerse the pre-section in 50%, 70%, 80%, and 90% ethanol successively once each, with an immersion time of 15 - 20 min each time; S92: Immerse the pre-section in 100% ethanol twice, with an immersion time of 5 - 10 min each time; S93: Immerse the pre-section in a 50% (v / v) xylene-ethanol solution for 15 - 20 min; S94: Immerse the pre-section in xylene twice, with an immersion time of 5 - 10 min each time; Seal the pre-section with neutral resin to obtain a gonad section of the Chinese soft-shelled turtle embryo.
8. The method for sampling the gonads of Chinese soft-shelled turtle embryos during the embryonic period and preparing tissue sections as claimed in claim 1, wherein In the step S6, the thickness of the section is 3 - 6 μm. The section also needs to be spread and baked to form a pre-section. The spreading is done by spreading the section with water at 42 - 46 °C. The baking step is as follows: After spreading, take out the section and place it on a glass slide, and place the glass slide with the section in an environment at 60 °C and dry for 8 - 12 min to obtain a pre-section.
9. The method for sampling the gonads and preparing tissue sections during the embryonic stage of the Chinese soft-shelled turtle as described in claim 1, wherein, In the step S6, the mold includes a base and a sample cup. The shape of the base fits the embedding machine fixture. The sample cup is a cylinder with a diameter of 0.3 - 1 cm and a height of 0.5 - 1.5 cm. The sample cup and the base are connected by threads. A pull ring for demolding is provided on the side of the sample cup.
10. A gonad section of the soft-shelled turtle embryo, characterized in that, Prepared by using the method for sampling and tissue section preparation of the gonad of the Chinese soft-shelled turtle embryo according to any one of claims 1 - 9.