A method for improving observation effect of aquatic animal tissue

Abstract

The present invention discloses a method for improving the observation effect of aquatic animal tissue, which belongs to the field of tissue observation. The present invention prepares slices of samples obtained after pre-treatment of aquatic animals with balance and anesthesia, which can effectively reduce the changes in tissue structure caused by the stress response of the aquatic animals themselves, obtain more accurate changes in the tissue structure of aquatic animals, obtain tissue slices with clear structures, and promote scientific development in the field of aquatic animal research. The prepared sea cucumber samples obtained after pre-treatment with balance and anesthesia can be directly used to prepare frozen slices, which greatly saves time and cost. The method provided by the present invention is simple to operate, has significant effects and low cost, and is easy to promote.

Description

Technical Field

[0001] The invention relates to a method for improving the observation effect of aquatic animal tissues, and belongs to the field of tissue observation. Background Art

[0002] Both sea cucumbers and sea urchins belong to the phylum Echinodermata. Sea cucumbers are high in protein, low in fat, and rich in essential amino acids, vitamins, essential fatty acids, and macro- and trace elements. Sea urchins are rich in eicosenoic acid, and its extract, bonelliine, has been shown to inhibit cancer cell growth. The high nutritional value of sea cucumbers is gaining increasing recognition, leading to increasing research on their processing characteristics. The sea cucumber body wall is primarily composed of collagen, intercalated with glycosaminoglycan bridges. The degradation of these structures under various processing conditions can lead to structural changes in the sea cucumber body wall, impacting its texture, nutritional content, and sensory quality. Accurately observing these changes in the sea cucumber body wall is crucial.

[0003] Currently, one method for observing the microstructure of aquatic products is optical microscopy, which requires the preparation of sample sections. Section staining techniques are essential in histological research and include frozen sections, paraffin sections, carbon wax sections, ultrathin sections, and plastic sections. Paraffin sections are often used to observe the microscopic structure of aquatic animals. Due to its advantages, such as minimal equipment requirements and long-term sample preservation, paraffin sections are widely used in conventional histological and pathological sections and also play a vital role in the study of aquatic product tissue structure. The traditional paraffin section preparation process typically involves fixing the sample in 10% diformaldehyde solution immediately after sampling, followed by a series of steps including dehydration, clearing, wax impregnation, embedding, and sectioning. This method, when used in live aquatic animals, can cause muscle fibers to contract and reassemble during the sampling and processing process, leading to a disordered collagen fiber structure, which can affect observation and make it difficult to obtain accurate and clear tissue structures. Summary of the Invention

[0004] The present invention primarily provides a method for improving the observation of aquatic animal tissue sections. The method reduces the stress response of aquatic animals during the sectioning process, effectively preserving the original microscopic structure of the sample. The method is also applied to observing aquatic animal tissue structures using paraffin sections, frozen sections, and scanning electron microscopes. The present invention aims to provide a method for improving the observation of aquatic animal tissue sections, thereby obtaining more accurate information on changes in aquatic animal tissue structure.

[0005] The present invention provides a method for improving the imaging clarity of aquatic animal tissue slices. The method comprises pre-treating the aquatic animal before preparing the tissue slices. The pre-treatment comprises treating the fresh aquatic animal with artificial seawater balancing solution and seawater anesthetic solution respectively.

[0006] In one embodiment, the pretreatment is to soak the fresh aquatic animals in artificial seawater balancing solution and seawater anesthetic solution for a certain period of time.

[0007] In one embodiment, the pretreatment is to immerse the fresh aquatic animal in artificial seawater balance solution for 0.5 to 2.5 hours, and then immerse it in seawater anesthetic solution for 0.5 to 2.5 hours.

[0008] In one embodiment, the artificial seawater balance solution is a 2-5% sodium chloride solution.

[0009] In one embodiment, the seawater anesthetic solution is a 2-5% sodium chloride solution containing 2-8% MgCl2.

[0010] In one embodiment, the seawater anesthetic solution is seawater anesthetic solution pre-cooled with ice water.

[0011] In one embodiment, the aquatic animal comprises the phylum Echinodermata.

[0012] In one embodiment, the aquatic animal comprises a sea cucumber or a sea urchin.

[0013] In one embodiment, the tissue sections include paraffin sections, frozen sections, carbon wax sections, ultrathin sections or plastic sections.

[0014] The present invention also provides a kit for improving the imaging clarity of aquatic animal tissue sections. The kit comprises an artificial seawater balancing solution and a seawater anesthetic solution. The artificial seawater balancing solution is a sodium chloride solution with a certain concentration, and the seawater anesthetic solution is a sodium chloride solution containing a certain concentration of magnesium chloride.

[0015] In one embodiment, the artificial seawater balance solution is a 2-5% sodium chloride solution.

[0016] In one embodiment, the seawater anesthetic solution is a 2-5% sodium chloride solution containing 2-8% MgCl2.

[0017] The present invention also provides application of the method or the kit in the field of section staining technology.

[0018] The present invention also provides application of the method or the kit in the field of aquatic animal histology research.

[0019] Beneficial effects of the present invention:

[0020] 1. Slicing of aquatic animal samples obtained after pre-treatment with balance and anesthesia can effectively reduce the changes in tissue structure caused by the stress response of the aquatic animals themselves, obtain more accurate changes in the tissue structure of sea cucumbers, obtain tissue slices with clear structure, and promote scientific development in the field of aquatic animal research.

[0021] 2. The prepared sea cucumber samples obtained after equilibration and anesthesia pretreatment can be directly used to prepare frozen sections. The collagen fibers of the sea cucumber body wall samples will not be damaged, and the orderly arranged collagen fibers and proteoglycans can be clearly observed. Compared with paraffin sections, the time cost is greatly saved. The method provided by the present invention is simple to operate, has significant effects, and is easy to promote.

[0022] 3. The paraffin sections prepared from the sea urchin samples obtained after equilibrium and anesthesia pretreatment can show the reticular structure and gametocytes composed of nutrient phagocytes more clearly than traditional paraffin sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Paraffin sections of the sea cucumber body wall observed under an optical microscope; Figure A shows the tissue structure of a fresh sea cucumber that has not been balanced and anesthetized, and Figure B shows the tissue structure of a fresh sea cucumber that has been balanced and anesthetized.

[0024] Figure 2 Frozen sections were observed under an optical microscope; Figure A shows the tissue structure of fresh sea cucumbers that have not been balanced and anesthetized, and Figure B shows the tissue structure of fresh sea cucumbers that have been balanced and anesthetized.

[0025] Figure 3 To observe the structure of mutable collagen tissue (MCT) in the cortex of the body wall of fresh sea cucumbers using a scanning electron microscope; Figure A shows the tissue structure of fresh sea cucumbers that have not been balanced and anesthetized, and Figure B shows the tissue structure of fresh sea cucumbers that have been balanced and anesthetized.

[0026] Figure 4 Fresh sea urchin gonad tissue sections were observed under an optical microscope; Figure A shows the gonad tissue structure of fresh sea urchin that has not been balanced and anesthetized, and Figure B shows the gonad tissue structure of fresh sea urchin that has been balanced and anesthetized. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] The experimental materials involved in the following examples are:

[0029] Weigert's iron hematoxylin was purchased from Beijing Leigen Biotechnology Co., Ltd., catalog number: DC0045. VG staining solution was purchased from Beijing Leigen Biotechnology Co., Ltd., catalog number: DC0046. Alcian blue staining solution was purchased from Beijing Leigen Biotechnology Co., Ltd., catalog number: DG0007. Periodic acid solution was purchased from Beijing Baoxidi Co., Ltd., catalog number: P815692-25g. Schiff reagent was purchased from Solabio, catalog number: G1286-100 mL.

[0030] Example 1: Preparation of paraffin sections

[0031] S1. Sampling and balancing: fresh sea cucumbers were immersed in artificial seawater balancing solution (3.5% sodium chloride solution) for 1 hour to obtain the equilibrated living sea cucumbers;

[0032] S2, anesthesia: immersing the living sea cucumber described in step S1 in ice-warm seawater anesthetic solution (artificial seawater balanced solution containing 3.5% MgCl2) for 1 hour to obtain the anesthetized living sea cucumber;

[0033] S3, cutting: Take the anesthetized living sea cucumber, cut it open along the abdomen, remove the teeth and internal organs, clean it, and place the sea cucumber body wall with the abdomen facing down, cut off the abdomen longitudinally, and cut off the head and tail horizontally. Lay the sea cucumber flat on its back and cut it into 0.5 cm x 0.5 cm x 0.5 cm sea cucumber body wall sample blocks;

[0034] S4. Fixation: Immerse the sea cucumber body wall sample block in Bouin's fixative (seawater saturated with picric acid: 40% formaldehyde solution: glacial acetic acid = 15:5:1) for 24 hours to obtain a fixed sea cucumber body wall sample block;

[0035] S5. Gradient Dehydration: The fixed sea cucumber body wall sample blocks were trimmed and placed in embedding cassettes for gradient dehydration in an automatic dehydrator. The dehydration program was set as follows: 50%, 60%, 70%, 80%, and 90% ethanol for 2 hours each, followed by 95% ethanol, 100% ethanol I, and 100% ethanol II for 1 hour each.

[0036] S6 transparency: The dehydrated sea cucumber body wall sample blocks were transparentized in a concentration gradient xylene in an automatic dehydrator (in the order of 1 / 3 concentration xylene, 1 / 2 concentration xylene, 2 / 3 concentration xylene, full xylene I, and full xylene II, respectively, for 40 min);

[0037] S7 wax immersion: immerse the transparent sea cucumber body wall sample block in a wax tank for 2.5 hours;

[0038] S8 Embedding: Place the wax-impregnated sea cucumber body wall sample block in a paraffin embedding machine and soak in paraffin for 10 minutes to complete the embedding. During the embedding process, ensure that the sea cucumber body wall is completely embedded in liquid paraffin and the position is fixed without tilting;

[0039] S9 Slicing, spreading, and scooping: After cooling the embedded sea cucumber body wall sample block, slice it with a paraffin slicer to a thickness of 6 μm. Place the sliced ​​sea cucumber body wall tissue slices in 43°C deionized water for spreading. Scoop out the spread sea cucumber body wall tissue using an adhesive slide.

[0040] S10 dewaxing: Immerse the adhered slides with sea cucumber body wall tissue in a complete xylene solution and heat in a 60°C oven for 30 minutes. After cooling to room temperature, soak them in complete xylene I solution for 10 minutes, complete xylene II solution for 8 minutes, 2 / 3 concentration xylene solution for 5 minutes, 1 / 2 concentration xylene solution for 5 minutes, and 1 / 3 concentration xylene solution for 5 minutes.

[0041] S11 Rehydration: Rehydrate with different concentrations of ethanol, soaking in 100% I, 100% II, 95%, 90%, 80%, 70%, 60%, and 50% ethanol for 3 minutes respectively, and finally soaking in distilled water for 5 minutes and then drying.

[0042] S12 staining: Use Weigert iron hematoxylin (hematoxylin A and B mixed in a 1:1 ratio for 2 minutes); wash thoroughly with tap water (approximately 10 minutes or more); use paper to absorb the moisture around the tissue, immerse in VG staining solution (solution A:solution B = 1:9) for staining for 5 minutes; then place the sections in anhydrous ethanol and xylene for 10 minutes each.

[0043] S13 Sealing: Use neutral resin to add drops on the treated slide to seal the slide, and then ventilate for 24 hours to make it a permanent slice.

[0044] Use an optical microscope to observe the slices. The principle of VG dye is that the basic amino acids in collagen fibers can combine with acidic dyes, which can show collagen fibers and other tissues. After VG staining, collagen fibers appear red and myofibrils appear yellow. Figure 1 As shown, the body wall of fresh sea cucumbers is primarily composed of collagen fibers. In Figure 1A, the density of the fibers is low, the interfiber spaces are large, and the fibers are distributed in a disordered manner. Compared to unbalanced and anesthetized sea cucumbers, the fibers in Figure 1B are arranged in a regular pattern and have a clear structure.

[0045] Example 2 Preparation of frozen sections

[0046] S1. Sampling and balancing: fresh sea cucumbers were immersed in artificial seawater balancing solution (3.5% sodium chloride solution) for 1 hour to obtain the equilibrated living sea cucumbers;

[0047] S2, anesthesia: immersing the living sea cucumber described in step S1 in ice-warm seawater anesthetic solution (artificial seawater balanced solution containing 3.5% MgCl2) for 1 hour to obtain the anesthetized living sea cucumber;

[0048] S3, cutting: Take the anesthetized living sea cucumber, cut it open along the abdomen, remove the teeth and internal organs, clean it, and place the sea cucumber body wall with the abdomen facing down, cut off the abdomen longitudinally, and cut off the head and tail horizontally. Lay the sea cucumber flat on its back and cut it into 0.5 cm x 0.5 cm x 0.5 cm sea cucumber body wall sample blocks;

[0049] S4, freezing the sea cucumber body wall sample block with a freezing microtome and cutting it into 10 μm slices;

[0050] S5. Staining: Stain the slices with Alcian blue staining solution for 14 minutes, then wash them with distilled water three times, 2 minutes each time; oxidize with periodic acid solution for 4 minutes; pour off the periodic acid solution and stain with Schiff reagent in the dark for 14 minutes; pour off the Schiff reagent and rinse gently with running water for 12 minutes; place the slices in ethanol and xylene for 10 minutes each; dry the slices and store them.

[0051] The sections were observed and photographed using an optical microscope.

[0052] The results are as follows Figure 2 As shown, the AB-PAS staining method specifically stains proteoglycans, resulting in blue staining of acidic polysaccharides such as proteoglycans and purple-red staining of collagen fibers. The sea cucumber body wall is primarily composed of collagen fibers connected by glycosaminoglycan bridges. Staining reveals that the purple-red collagen fibers (Figure 2A) are partially broken, the number of blue proteoglycan sites is reduced, and the glycosaminoglycan bridges are slightly broken, resulting in an unevenly distributed cross-linked network. Compared to unequilibrated and unanesthetized sea cucumbers, the purple-red collagen fibers (Figure 2B) are orderly arranged, with evenly arranged blue proteoglycans present between the collagen fibers, forming a tight network with the purple-red collagen fibers.

[0053] Example 3

[0054] S1. Sampling and balancing: fresh sea cucumbers were immersed in artificial seawater balancing solution (3.5% sodium chloride solution) for 1 hour to obtain the equilibrated living sea cucumbers;

[0055] S2, anesthesia: immersing the living sea cucumber described in step S1 in ice-warm seawater anesthetic solution (artificial seawater balanced solution containing 3.5% MgCl2) for 1 hour to obtain the anesthetized living sea cucumber;

[0056] S3, cutting: Take the anesthetized living sea cucumber, cut it open along the abdomen, remove the teeth and internal organs, clean it, and place the sea cucumber body wall with the abdomen facing down, cut off the abdomen longitudinally, and cut off the head and tail horizontally. Lay the sea cucumber flat on its back and cut it into 0.5 cm x 0.5 cm x 0.5 cm sea cucumber body wall sample blocks;

[0057] S4. Fix the sea cucumber body wall specimens in 2.5% glutaraldehyde solution at 4°C for 24 h. Rinse with deionized water and dehydrate in gradient ethanol solutions (50%, 70%, 90%, 95%, and 100%) for 15 min each. Freeze-dry the dehydrated tissue blocks. Fragment the freeze-dried specimens at low temperatures and place them on a specimen stage for gold coating. The microstructure of the sea cucumber body wall was observed under a scanning electron microscope.

[0058] The results are as follows Figure 3 As shown in Figure 3A, scanning electron microscopy was used to observe the structure of the mutable collagen tissue (MCT) in the cortex of the body wall of fresh sea cucumbers. In Figure 3A, the interfiber spaces are blurred and disorganized, the collagen fiber surface is roughened, and small fiber bundles shrink and aggregate into large bundles. Compared with Figure 3A, Figure 3B shows that the collagen fibers in the fresh sea cucumber MCT structure are complete fiber bundles, with dense and orderly fibers and a certain degree of directionality.

[0059] Example 4

[0060] The sea urchin gonad is composed of individual granules filled with phagocytes and gametes. The primary protein component of the phagocytes is sea urchin yolk protein. Observing the microstructural distribution of phagocytes and gametes can more accurately assess changes in the sea urchin gonad's histological structure. The effectiveness of the balancing and anesthesia procedures used in the preparation of sea urchin gonadal tissue sections was verified.

[0061] S1. Sampling and balancing: fresh sea urchins were soaked in artificial seawater balancing solution (3.5% sodium chloride solution) for 1 hour to obtain the equilibrated living sea urchins;

[0062] S2. Anesthesia: Immerse the living sea urchin described in step S1 in ice-warm seawater anesthetic solution (balanced artificial seawater solution containing 7.5% MgCl2) for 1 hour to obtain an anesthetized living sea urchin;

[0063] S3. Fixation: Anesthetize a live sea urchin and place it on ice. Open the shell of the sea urchin and remove the gonad. Immerse the sea urchin in a 10% neutral formalin solution (0.01 mol / L neutral phosphate buffer: 40% formaldehyde solution = 9:1) for 48 h to obtain a fixed sea urchin gonad sample block.

[0064] S4. Gradient Dehydration: Place the trimmed sea urchin gonadal specimens into an embedding cassette and perform gradient dehydration in an automatic dehydrator. The dehydration program is set as follows: 50%, 60%, 70%, 80%, and 90% ethanol for 30 minutes each, followed by 95% ethanol, 100% ethanol I, and 100% ethanol II for 1 hour each.

[0065] S5. Transparent: The dehydrated sea urchin gonad sample block was transparentized in a concentration gradient xylene in an automatic dehydrator (transparentization in the order of 1 / 3 concentration xylene, 1 / 2 concentration xylene, 2 / 3 concentration xylene, full xylene I, and full xylene II for 40 min each);

[0066] S6. Wax immersion: immerse the transparent sea urchin gonad sample block in a wax tank for 2.5 hours;

[0067] S7. Embedding: Place the wax-impregnated sea urchin gonad sample block in a paraffin embedding machine and embed it in paraffin for 10 minutes. During the embedding process, ensure that the sea urchin body wall is completely embedded in the liquid paraffin and is fixed in position without tilting.

[0068] S8. Slice, spread, and retrieve: After cooling the embedded sea urchin gonad sample block, slice it using a paraffin microtome, aligning the sea urchin particles with the blade. Slice to a thickness of 7 μm. Spread the sliced ​​sea urchin gonad tissue in 43°C deionized water. Retrieve the unfolded sea urchin gonad tissue using an adhesive slide.

[0069] S9. Dewaxing: Immerse the adhered slides with sea urchin gonadal tissue in a complete xylene solution and heat in a 60°C oven for 30 min. After cooling to room temperature, soak them in complete xylene I solution for 10 min, complete xylene II solution for 8 min, 2 / 3 xylene solution for 5 min, 1 / 2 xylene solution for 5 min, and 1 / 3 xylene solution for 5 min.

[0070] S10, rehydration: rehydrate with different concentrations of ethanol, in order, soak in 100% I, 100% II, 95%, 90%, 80%, 70%, 60%, and 50% ethanol for 3 minutes respectively, and finally soak in distilled water for 5 minutes and then dry;

[0071] S11. Staining: Use Weigert iron hematoxylin (hematoxylin A and B mixed in a 1:1 ratio for 2 minutes); wash thoroughly with tap water (approximately 10 minutes or more); dry the tissue with kitchen paper and stain with AB-PAS (Alcian blue-periodic acid-Schiff) staining solution for 5 minutes. Wash with distilled water for 1 minute, oxidize with periodic acid solution for 5 minutes, wash again with distilled water for 1 minute, and stain with Schiff's reagent for 5 minutes. Then, place the sections in anhydrous ethanol and xylene for 10 minutes each.

[0072] S12. Sealing: Use neutral resin to add drops on the treated slide to seal the slide, and then ventilate for 24 hours to make it a permanent slice.

[0073] Figure 4 Fresh sea urchin gonadal tissue sections were stained with Alcian blue-periodic acid Schiff staining (AB-PAS) using paraffin sections, and their microstructures were observed using an optical microscope. The sea urchin gonadal tissue consists of two parts: nutrient phagocytes (NP) and gametocytes. AB-PAS staining showed that nutrient phagocytes contain eosinophilic droplets and are stained blue, while gametocytes lack eosinophilic droplets and are stained purple or red. Figure 4 It can be seen that in both Figure 4A and Figure 4B, the gametocytes appear red and are surrounded by blue nutrient phagocytes. However, compared with Figure 4A, the network structure formed by the nutrient phagocytes and the gametocytes in Figure 4B are clearer.

[0074] In summary, the balancing and anesthesia treatment of fresh sea cucumbers and sea urchins performed by the present invention can effectively reduce the stress response of sea cucumber body wall tissue or sea urchin gonad tissue during slicing, and can more accurately observe changes in tissue structure.

[0075] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for improving the imaging clarity of aquatic animal tissue sections, characterized in that: The method comprises pre-treating aquatic animals before preparing tissue sections, wherein the pre-treatment comprises soaking the fresh aquatic animals in artificial seawater balancing solution and seawater anesthetic solution for a certain period of time; The pretreatment is to immerse the fresh aquatic animals in artificial seawater balance solution for 0.5 to 2.5 hours, and then immerse them in seawater anesthetic solution for 0.5 to 2.5 hours; The artificial seawater balance solution is a 2-5% sodium chloride solution, and the seawater anesthetic solution is a 2-5% sodium chloride solution containing 2-8% magnesium chloride.

2. The method according to claim 1, characterized in that The aquatic animals include the phylum Echinodermata.

3. The method according to claim 1, characterized in that The tissue sections include paraffin sections, frozen sections, carbon wax sections, ultrathin sections or plastic sections.

4. A kit for improving the imaging clarity of aquatic animal tissue sections, characterized in that: The kit comprises an artificial seawater balancing solution and a seawater anesthetic solution. The artificial seawater balancing solution is a sodium chloride solution of a certain concentration, and the seawater anesthetic solution is a sodium chloride solution containing a magnesium chloride solution of a certain concentration. The artificial seawater balancing solution is a 2-5% sodium chloride solution, and the seawater anesthetic solution is a 2-5% sodium chloride solution containing 2-8% magnesium chloride.

5. Use of the method according to any one of claims 1 to 3 or the kit according to claim 4 in the field of section staining technology.

6. Use of the method according to any one of claims 1 to 3 or the kit according to claim 4 in the field of aquatic animal histology research.

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