Method for in-vivo detection of fish adipocyte hyperplasia
By adding EDUs to fish feed and performing immune fluorescent staining, the lack of the lack of effects in the existing technology to detect the hyperplasia of fish fat cells in the body, realize the detection effect of high accuracy and high success rate, and provide fish breeding for fish breeding Important health monitoring methods.
Patent Information
- Application Number
- CN202411241023.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art lacks effective in-vivo detection methods to detect the proliferation of fish adipocytes, which limits the development of the breeding industry.
By mixing EDU as a mobilizine deoxyucleosides into fish feed, after feeding grass carp and bass, the adipose tissue is collected for immunofluorescence chromatics and image analysis, which clearly shows the hyperplasia of fat cells.
It has achieved high-precision and high-success rate in vivo detection of fish fat cell hyperplasia, provided theoretical basis and data support, and provided an important health monitoring method for fish farming.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of cell biotechnology, and particularly relates to a method for detecting fish adipocyte hyperplasia in vivo. Background Art
[0002] Grass carp and sea bass are representative herbivorous and carnivorous economic farmed fish in my country. They are tender and delicious, nutritious, rich in unsaturated fatty acids and a variety of trace elements. They are widely distributed in rivers, lakes and reservoirs in my country and are deeply loved by consumers. However, in order to save feed feeding costs, high-energy feed has been widely used in the breeding process, which has led to a decrease in grass carp's ability to utilize lipids and excessive accumulation, seriously affecting the health of the fish. Adipose tissue is the most important lipid storage site in animals and is specifically used to store excess energy. Studies in fish and mammals have shown that under conditions of excess energy, increasing the number of fat cells in adipose tissue (promoting fat cell proliferation) helps healthy adipose tissue expand and maintain metabolic homeostasis, which has a positive effect on the body. Fat cell proliferation is essential for the health of the body, and studying fat cell proliferation is very necessary to solve the health problems of grass carp. However, little is known about how to detect the proliferation of fat cells in the fat tissue of grass carp, which to some extent limits the development of the aquaculture industry. Although there are many methods that can be used to detect cell proliferation in vitro, there are certain differences between in vivo and in vitro. In vitro cannot fully reflect the complex situation in vivo. So far, there is no systematic, feasible and successful method for detecting the proliferation of fish fat cells in vivo. Therefore, it is urgent to establish a method for detecting the proliferation of fat cells in vivo. 5-ethynyl-2′-deoxyuridine (EdU) is a new thymidine deoxynucleoside analog that can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis. Subsequently, EdU-positive cells are newly proliferated cells by immunofluorescence staining. At the same time, if it is co-stained with other cell markers, the type of proliferating cells can be determined, and quantitative analysis can also be performed. In addition, EdU is now commonly used to replace 5-bromo-2′-deoxyuridine (BrdU) and is used to detect cell proliferation. On the one hand, EdU has simpler operation steps, fewer influencing factors, and better stability than BrdU. Since BrdU requires the use of specific antibodies, it is easy to interfere with other target proteins when performing multiple labels and is cumbersome. On the other hand, EdU does not require the use of antibodies, is easy to operate and sensitive, and only requires simple reactions. It is a new substance and a new method for detecting cell proliferation that is superior to BrdU. However, most of the current uses of EdU are only in in vitro cells, and there are no reports on how to use EdU in fish and detect cell proliferation, especially in fish. Summary of the invention
[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for detecting fish adipocyte hyperplasia in vivo, which belongs to the field of cell biotechnology. The method is as follows: EdU, a thymidine deoxynucleoside analog based on the DNA synthesis process, is mixed into the feed and fed to grass carp and sea bass for 3 days, and then any feed is used for feeding for 2 weeks, and then the grass carp and sea bass adipose tissue samples are collected for fixation, preparation and immunofluorescence staining, and then the image is collected and processed and analyzed using a fluorescence microscope. The image results can clearly show the green adipocyte membrane and the blue cell nucleus and the red EdU new cell nucleus, wherein the newly generated adipocyte nucleus becomes purple after superposition. The method in the present invention is convenient, fast, high-precision and high-success rate, and provides a theoretical basis and data support for the in vivo detection of fish adipocyte hyperplasia.
[0004] Technical solution: A method for detecting fish adipocyte hyperplasia in vivo, comprising the following steps: S1. Weighing EdU and other feed ingredients, adding water to mix, granulating and drying to prepare fish feed containing EdU; S2. Fish were fed with EdU-containing feed for 3 days, and then fed with any feed for 2 weeks before collecting fish adipose tissue samples; S3. Fixing and slicing the fish fat tissue to obtain a fish fat tissue test sample; S4. Immunofluorescence staining and image analysis of fish adipose tissue samples. Furthermore, the fish is a fish containing abdominal adipose tissue. Furthermore, the fish containing abdominal adipose tissue include grass carp and seabass. Furthermore, the amount of EdU added in step S1 is 3-5 mg / kg. Furthermore, the other feed ingredients in step S1 include other grass carp feed ingredients and other seabass feed ingredients. Furthermore, other ingredients of the grass carp feed are: casein 35%, gelatin 8.75%, corn starch 30-50%, soybean oil 4-10%, choline chloride 0.5%, premix 1%, bentonite 20.65% and 16.65%, BHT 0.01%, based on the total weight of the feed. Furthermore, other ingredients of the sea bass feed are: fish meal 35%, soybean meal 4%, chicken meal 14%, soy protein isolate 8%, cottonseed protein 8%, corn starch 8%, fish oil 9.22%, monocalcium phosphate 1%, premix 1%, choline chloride 0.5%, vitamin C 0.1%, DL-methionine 0.5%, antioxidant 0.1%, and bentonite 10.58%, based on the total weight of the feed. Furthermore, the amount of water added in step S1 is 40% of the total weight of the feed components, the adding method is a small amount and multiple times in a mixer, and the granulation equipment is a ring die granulator with a diameter of 4 mm. Furthermore, the immunofluorescence staining method in step S4 comprises the following steps: Step 1. Dewaxing, repairing and sealing of fish fat tissue test samples; Step 2. Perform EdU staining to obtain stained sections; Step 3. Add Caveolin-1 antibody to the stained sections and incubate overnight at 4°C. Step 4. Add FITC fluorescent secondary antibody, incubate at room temperature for 2 hours, then stain the nucleus and seal the slices to prepare fish fat cell detection sections. Furthermore, the image analysis method in step S4 comprises the following steps: Step 1. Observe the fish adipocyte detection slices under a fluorescence microscope and collect images; Step 2. The images were processed and analyzed using ImageJ software. Beneficial effects: 1. The present invention adds EdU to the feed fed to grass carp and seabass. EdU, as a new thymidine deoxynucleoside analog, can replace thymidine in the DNA synthesis process of grass carp and seabass adipocytes and be integrated into the newly generated adipocyte DNA. Based on this principle, after immunofluorescence staining of the test sample, the hyperplasia changes of grass carp and seabass adipocytes can be clearly detected, and it can be effectively used for in vivo detection of adipocyte hyperplasia of grass carp and seabass. 2. The method for in vivo detection of fish adipocyte hyperplasia provided by the present invention has the characteristics of high success rate, high accuracy, high precision and simple operation. It can meet the needs of studying in vivo cell hyperplasia in fish and has good application prospects and research value. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the food intake of grass carp during two weeks after taking EdU in Example 1, Comparative Example 1 and Comparative Example 2; Figure 2 is the food intake of seabass during two weeks after taking EdU in Example 2, Comparative Example 3 and Comparative Example 4; Figure 3 This is an immunofluorescence staining image of EdU, DAPI, and Caveolin-1 in adipose tissue of grass carp after two weeks of EdU ingestion in Example 3; Figure 4 This is an immunofluorescence staining image of EdU, DAPI, and Caveolin-1 in adipose tissue of seabass after two weeks of EdU ingestion in Example 4; Figure 5is the immunofluorescence staining of EdU, DAPI and Caveolin-1 in the adipose tissue of grass carp after two weeks of EdU ingestion in comparative example 5; Figure 6 is the immunofluorescence staining of EdU, DAPI and Caveolin-1 in the adipose tissue of seabass after two weeks of EdU intake in comparative example 6; Figure 7 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1 A method for preparing a grass carp feed containing EdU comprises the following steps Step 1. Weigh 1050 g casein, 262.5 g gelatin, 900 g corn starch, 120 g soybean oil, 15 g choline chloride, 30 g premix, 619.5 g bentonite, 499.5 g, 0.3 g BHT, and 9 mg EdU, mix well to obtain a mixed raw material; Step 2. The mixed raw materials are put into a mixer, and 1.2 L of water is added to the mixer in small amounts and multiple times to mix well to obtain a granulation base material; Step 3. Pellet the base material into pellet feed using a ring die pelletizer with a diameter of 4 mm; Step 4. After the pellet feed is dried, it is stored at -20°C to prepare the grass carp feed containing EdU. Example 2 A method for preparing a seabass feed containing EdU comprises the following steps: Step 1. Weigh 1050 g fish meal, 120 g soybean meal, 420 g chicken meal, 240 g soy protein isolate, 240 g cottonseed protein, 240 g corn starch, 276.6 g fish oil, 30 g calcium dihydrogen phosphate, 30 g premix, 15 g choline chloride, 3 g vitamin C, 15 g DL-methionine 0.5%, 3 g antioxidant, 317.4 g bentonite, and 9 mg EdU, mix well, and obtain a mixed raw material; Step 2. The mixed raw materials are put into a mixer, and 1.2 L of water is added to the mixer in small amounts and multiple times to mix well to obtain a granulation base material; Step 3. Pellet the base material into pellet feed using a ring die pelletizer with a diameter of 4 mm; Step 4. The pellet feed is dried and stored at -20°C to prepare EdU-containing sea bass feed. Example 3 A method for detecting grass carp adipocyte hyperplasia in vivo comprises the following steps: S1. Feeding grass carp with the EdU-containing grass carp feed prepared in Example 1 for 3 days, and then feeding with normal grass carp feed for 2 weeks, and then collecting grass carp adipose tissue samples; S2. Fixing and slicing the grass carp adipose tissue to obtain a grass carp adipose tissue test sample; S3. Grass carp adipose tissue test samples were first dewaxed in xylene solution for 20 min (dewaxed twice), then dewaxed in anhydrous ethanol solution for 5 min (dewaxed twice), then dewaxed in 75% ethanol solution for 5 min, and finally washed with tap water; S4. Place in a pressure cooker containing pH 6.0 citric acid antigen retrieval solution for antigen retrieval, with the jet timer set at 2.5 min. After natural cooling, place in pH 7.4 PBS buffer, and wash on a decolorizing shaker 3 times, 5 min each time. S5. After drying, draw a circle around the tissue with a tissue pen (to prevent the antibody from flowing away), add 5% BSA in the circle, and block at room temperature for 1 hour; S6. After shaking off the blocking solution, add Apollo staining reaction solution, incubate on a decolorizing shaker for 30 minutes under light-proof room temperature conditions, discard the staining reaction solution, and then add a penetrant (0.5% TritonX-100 in PBS solution) to wash on a decolorizing shaker 2-3 times, each time for 10 minutes, and finally add methanol to wash 1-2 times, each time for 5 minutes, wash with PBS buffer for 5 minutes, and after 30 minutes, wash with PBS buffer for 10 minutes (wash 3 times) to obtain grass carp adipocyte stained sections; S7. Add Caveolin-1 antibody to the grass carp adipocyte staining sections, place them flat in a humidified box, and incubate them overnight at 4°C. (Add a small amount of water to the humidified box to prevent the antibody from evaporating.) S8. Place in PBS buffer at pH 7.4, shake and wash on a decolorizing shaker for 3 times, 5 min each time, then add FITC fluorescent secondary antibody to cover the tissue, and incubate at room temperature for 2 h; S9. Add DAPI, incubate at room temperature for 10 min, then wash in PBS buffer for 10 min (wash 3 times), and then seal with anti-fluorescence quencher; S10. Observe and collect images under a fluorescence microscope, and use ImageJ software for image processing and analysis. Example 4 A method for detecting seabass adipocyte hyperplasia in vivo comprises the following steps: S1. Feeding perch with the EdU-containing perch feed prepared in Example 2 for 3 days, and then feeding with normal perch feed for 2 weeks, collecting perch adipose tissue samples; S2. fixing and slicing the seabass adipose tissue to obtain a seabass adipose tissue test sample; S3. The seabass adipose tissue test samples were first dewaxed in a xylene solution for 20 min (dewaxed twice), then dewaxed in an absolute ethanol solution for 5 min (dewaxed twice), then dewaxed in a 75% ethanol solution for 5 min, and finally washed with tap water; S4. Place in a pressure cooker containing pH 6.0 citric acid antigen retrieval solution for antigen retrieval, with the jet timer set at 2.5 min. After natural cooling, place in pH 7.4 PBS buffer, and wash on a decolorizing shaker 3 times, 5 min each time. S5. After drying, draw a circle around the tissue with a tissue pen (to prevent the antibody from flowing away), add 5% BSA in the circle, and block at room temperature for 1 hour; S6. After shaking off the blocking solution, add Apollo staining reaction solution, incubate on a decolorizing shaker for 30 minutes under light-proof room temperature conditions, discard the staining reaction solution, and then add a penetrant (0.5% TritonX-100 in PBS solution) to wash on a decolorizing shaker 2-3 times, each time for 10 minutes, and finally add methanol to wash 1-2 times, each time for 5 minutes, wash with PBS buffer for 5 minutes, and after 30 minutes, wash with PBS buffer for 10 minutes (wash 3 times) to obtain sea bass adipocyte staining sections; S7. First, add Caveolin-1 antibody to the perch adipocyte staining sections, place them flat in a humidified box, and incubate them overnight at 4°C. (Add a small amount of water to the humidified box to prevent the antibody from evaporating.) S8. Place in PBS buffer at pH 7.4, shake and wash on a decolorizing shaker for 3 times, 5 min each time, then add FITC fluorescent secondary antibody to cover the tissue, and incubate at room temperature for 2 h; S9. Add DAPI, incubate at room temperature for 10 min, then wash in PBS buffer for 10 min (wash 3 times), and then seal with anti-fluorescence quencher; S10. Observe and collect images under a fluorescence microscope, and use ImageJ software for image processing and analysis. Comparative Example 1 The difference between this comparative example and Example 1 is that the amount of EdU added is 1 mg / kg. A method for preparing a grass carp feed containing EdU comprises the following steps: Step 1. Weigh 1050 g casein, 262.5 g gelatin, 900 g corn starch, 120 g soybean oil, 15 g choline chloride, 30 g premix, 619.5 g bentonite, 499.5 g, 0.3 g BHT, and 3 mg EdU, mix well to obtain a mixed raw material; Step 2. The mixed raw materials are put into a mixer, and 1.2 L of water is added in small amounts and multiple times to mix well to obtain a granulation base material; Step 3. Pellet the base material into pellet feed using a ring die pelletizer with a diameter of 4 mm; Step 4. After the pellet feed is dried, it is stored at -20°C to prepare the grass carp feed containing EdU. Comparative Example 2 The difference between this comparative example and Example 1 is that the added amount of EdU is 7 mg / kg. A method for preparing a grass carp feed containing EdU comprises the following steps: Step 1. Weigh 1050g casein, 262.5g gelatin, 900g corn starch, 120g soybean oil, 15g choline chloride, 30g premix, 619.5g bentonite and 499.5g, 0.3g BHT, 21mg EdU, mix them evenly to obtain a mixed raw material; Step 2. Put the mixed raw materials into a mixer, add 1.2L of water and add it to the mixer in small amounts and multiple times to mix, to obtain a granulation base; Step 3. Pellet the base material into pellet feed using a ring die pelletizer with a diameter of 4 mm; Step 4. After the pellet feed is dried, it is stored at -20°C to prepare the grass carp feed containing EdU. Comparative Example 3 The difference between this comparative example and Example 2 is that the added amount of EdU is 1 mg / kg. A method for preparing a seabass feed containing EdU comprises the following steps: Step 1. Weigh 1050 g fish meal, 120 g soybean meal, 420 g chicken meal, 240 g soy protein isolate, 240 g cottonseed protein, 240 g corn starch, 276.6 g fish oil, 30 g calcium dihydrogen phosphate, 30 g premix, 15 g choline chloride, 3 g vitamin C, 15 g DL-methionine 0.5%, 3 g antioxidant, 317.4 g bentonite, and 3 mg EdU, mix well, and obtain a mixed raw material; Step 2. The mixed raw materials are put into a mixer, and 1.2 L of water is added to the mixer in small amounts and multiple times to mix well to obtain a granulation base material; Step 3. Pellet the base material into pellet feed using a ring die pelletizer with a diameter of 4 mm; Step 4. The pellet feed is dried and stored at -20°C to prepare EdU-containing sea bass feed. Comparative Example 4 The difference between this comparative example and Example 2 is that the amount of EdU added is 7 mg / kg. A method for preparing a seabass feed containing EdU comprises the following steps: Step 1. Weigh 1050 g fish meal, 120 g soybean meal, 420 g chicken meal, 240 g soy protein isolate, 240 g cottonseed protein, 240 g corn starch, 276.6 g fish oil, 30 g calcium dihydrogen phosphate, 30 g premix, 15 g choline chloride, 3 g vitamin C, 15 g DL-methionine 0.5%, 3 g antioxidant, 317.4 g bentonite, and 21 mg EdU, mix well, and obtain a mixed raw material; Step 2. The mixed raw materials are put into a mixer, and 1.2 L of water is added to the mixer in small amounts and multiple times to mix well to obtain a granulation base material; Step 3. Pellet the base material into pellet feed using a ring die pelletizer with a diameter of 4 mm; Step 4. The pellet feed is dried and stored at -20°C to prepare EdU-containing sea bass feed. Comparative Example 5 The difference between this comparative example and Example 3 is that the grass carp feed containing EdU is prepared by Comparative Example 1. A method for detecting grass carp adipocyte hyperplasia in vivo comprises the following steps: S1. The grass carp was fed with the EdU-containing grass carp feed prepared in Comparative Example 1 for 3 days, and then adipose tissue samples of the grass carp were collected after feeding with normal grass carp feed for 2 weeks; S2. Fixing and slicing the grass carp adipose tissue to obtain a grass carp adipose tissue test sample; S3. Grass carp adipose tissue test samples were first dewaxed in xylene solution for 20 min (dewaxed twice), then dewaxed in anhydrous ethanol solution for 5 min (dewaxed twice), then dewaxed in 75% ethanol solution for 5 min, and finally washed with tap water; S4. Place in a pressure cooker containing pH 6.0 citric acid antigen retrieval solution for antigen retrieval, with the jet timer set at 2.5 min. After natural cooling, place in pH 7.4 PBS buffer, and wash on a decolorizing shaker 3 times, 5 min each time. S5. After drying, draw a circle around the tissue with a tissue pen (to prevent the antibody from flowing away), add 5% BSA in the circle, and block at room temperature for 1 hour; S6. After shaking off the blocking solution, add Apollo staining reaction solution, incubate on a decolorizing shaker for 30 minutes under light-proof room temperature conditions, discard the staining reaction solution, and then add a penetrant (0.5% TritonX-100 in PBS solution) to wash on a decolorizing shaker 2-3 times, each time for 10 minutes, and finally add methanol to wash 1-2 times, each time for 5 minutes, wash with PBS buffer for 5 minutes, and after 30 minutes, wash with PBS buffer for 10 minutes (wash 3 times) to obtain grass carp adipocyte stained sections; S7. Add Caveolin-1 antibody to the grass carp adipocyte staining sections, place them flat in a humidified box, and incubate them overnight at 4°C. (Add a small amount of water to the humidified box to prevent the antibody from evaporating.) S8. Place in PBS buffer at pH 7.4, shake and wash on a decolorizing shaker for 3 times, 5 min each time, then add FITC fluorescent secondary antibody to cover the tissue, and incubate at room temperature for 2 h; S9. Add DAPI, incubate at room temperature for 10 min, then wash in PBS buffer for 10 min (wash 3 times), and then seal with anti-fluorescence quencher; S10. Observe and collect images under a fluorescence microscope, and use ImageJ software for image processing and analysis. Comparative Example 6 The difference between this comparative example and Example 4 is that the grass carp feed containing EdU is prepared by Comparative Example 3. A method for detecting seabass adipocyte hyperplasia in vivo comprises the following steps: S1. Feeding perch with EdU-containing perch feed prepared in Comparative Example 3 for 3 days, followed by feeding with normal perch feed for 2 weeks and collecting perch adipose tissue samples; S2. fixing and slicing the seabass adipose tissue to obtain a seabass adipose tissue test sample; S3. The seabass adipose tissue test samples were first dewaxed in a xylene solution for 20 min (dewaxed twice), then dewaxed in an absolute ethanol solution for 5 min (dewaxed twice), then dewaxed in a 75% ethanol solution for 5 min, and finally washed with tap water; S4. Place in a pressure cooker containing pH 6.0 citric acid antigen retrieval solution for antigen retrieval, with the jet timer set at 2.5 min. After natural cooling, place in pH 7.4 PBS buffer, and wash on a decolorizing shaker 3 times, 5 min each time. S5. After drying, draw a circle around the tissue with a tissue pen (to prevent the antibody from flowing away), add 5% BSA in the circle, and block at room temperature for 1 hour; S6. After shaking off the blocking solution, add Apollo staining reaction solution, incubate on a decolorizing shaker for 30 minutes under light-proof room temperature conditions, discard the staining reaction solution, and then add a penetrant (0.5% TritonX-100 in PBS solution) to wash on a decolorizing shaker 2-3 times, each time for 10 minutes, and finally add methanol to wash 1-2 times, each time for 5 minutes, wash with PBS buffer for 5 minutes, and after 30 minutes, wash with PBS buffer for 10 minutes (wash 3 times) to obtain sea bass adipocyte staining sections; S7. First, add Caveolin-1 antibody to the perch adipocyte staining sections, place them flat in a humidified box, and incubate them overnight at 4°C. (Add a small amount of water to the humidified box to prevent the antibody from evaporating.) S8. Place in PBS buffer at pH 7.4, shake and wash on a decolorizing shaker for 3 times, 5 min each time, then add FITC fluorescent secondary antibody to cover the tissue, and incubate at room temperature for 2 h; S9. Add DAPI, incubate at room temperature for 10 min, then wash in PBS buffer for 10 min (wash 3 times), and then seal with anti-fluorescence quencher; S10. Observe and collect images under a fluorescence microscope, and use ImageJ software for image processing and analysis. Performance Testing: (1) Feed intake The grass carp was fed with the EdU-containing grass carp feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 for 3 days, and then fed with normal grass carp feed for two weeks, and the feed intake of the grass carp was recorded every day; the sea bass was fed with the EdU-containing sea bass feed prepared in Example 2, Comparative Example 3 and Comparative Example 4 for 3 days, and then fed with normal sea bass feed for two weeks, and the feed intake of the sea bass was recorded every day. Depend on Figure 1 and Figure 2 It can be seen that the common difference between the EdU-containing grass carp feed prepared in Example 1, Comparative Example 1 and Comparative Example 2 and the EdU-containing sea bass feed prepared in Example 2, Comparative Example 3 and Comparative Example 4 is that the amount of EdU added is different, wherein the amount of EdU added in Example 1 and Example 2 is 3 mg / kg, the amount of EdU added in Comparative Example 1 and Comparative Example 3 is 1 mg / kg, and the amount of EdU added in Comparative Example 2 and Comparative Example 4 is 7 mg / kg. The grass carp and sea bass were fed with EdU-containing feed for 3 days, and then fed with normal feed for 2 weeks. The feed intake of Comparative Example 2 and Comparative Example 4 was significantly reduced, indicating that excessive EdU content will greatly reduce the feed intake of grass carp and sea bass, thereby blocking the growth of grass carp and sea bass, and further affecting the in vivo detection of fat cells of grass carp and sea bass. (2) Adipocyte proliferation and EdU positive cell rate ImageJ was used to analyze and overlay the three light source images in Example 3, Example 4, Comparative Example 5 and Comparative Example 6, and the EdU positive cell rate was counted, where DAPI: emits blue light, FITC: emits green light, and EdU: emits red light. Depend on Figure 3 and Figure 4 It can be seen that DAPI: cell nucleus emits blue light, FITC: adipocyte membrane emits green light, and EdU: newborn cells emit red light. After ImageJ superimposes the images of the three light sources, the purple fluorescence inside the green adipocytes is the newborn adipocytes. Figure 5 and Figure 6 It can be seen that no EdU positive cell rate was detected in Comparative Examples 5 and 6. The difference between them is that the amount of EdU added in the feed for grass carp and sea bass in the early stage was 1 mg / kg, which means that too little EdU content was added, which would result in less EdU integrated into the DNA of newly generated fat cells, and the fluorescent signal formed after immunofluorescence staining would be weaker, thus not enough to be detected. Depend on Figure 7 It can be seen that when grass carp is fed with EdU-containing feed, the EdU-positive cell rate of fat tissue of grass carp is 7.642% within 2 weeks, that is, the adipocyte proliferation rate (newly generated adipocytes) is 7.642%; when grass carp is fed with EdU-containing feed, the EdU-positive cell rate of fat tissue of sea bass is 10.322% within 2 weeks, that is, the adipocyte proliferation rate (newly generated adipocytes) is 10.322%. This shows that the method provided by the present invention has obvious staining effect, is simple and convenient, has a high success rate, and has a good application prospect in the detection of cell proliferation in fish in vivo. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.
Claims
1. A method for detecting fish adipocyte hyperplasia in vivo, characterized in that: The following steps are involved: S1. Weighing EdU and other feed ingredients, adding water to mix, granulating and drying to obtain fish feed containing EdU; S2. Fish were fed with EdU-containing feed for 3 days, and then fed with any feed for 2 weeks before collecting fish adipose tissue samples; S3. Fixing and slicing the fish fat tissue to obtain a fish fat tissue test sample; S4. Immunofluorescence staining and image analysis of fish adipose tissue samples.
2. A method for detecting fish adipocyte hyperplasia in vivo according to claim 1, characterized in that: The fish is a fish containing abdominal fat tissue.
3. A method for detecting fish adipocyte hyperplasia in vivo according to claim 2, characterized in that: The fish containing abdominal adipose tissue include grass carp and seabass.
4. The method for detecting fish adipocyte hyperplasia in vivo according to claim 1, characterized in that: The amount of EdU added in step S1 is 3-5 mg / kg.
5. The method for detecting fish adipocyte hyperplasia in vivo according to claim 1, characterized in that: The other feed ingredients in step S1 include other grass carp feed ingredients and other seabass feed ingredients.
6. The method for detecting fish adipocyte hyperplasia in vivo according to claim 5, characterized in that: Other ingredients of grass carp feed are: casein 35%, gelatin 8.75%, corn starch 30-50%, soybean oil 4-10%, choline chloride 0.5%, premix 1%, bentonite 20.65% and 16.65%, BHT 0.01%, based on the total weight of the feed.
7. The method for detecting fish adipocyte hyperplasia in vivo according to claim 5, characterized in that: Other ingredients of sea bass feed are: fish meal 35%, soybean meal 4%, chicken meal 14%, soy protein isolate 8%, cottonseed protein 8%, corn starch 8%, fish oil 9.22%, monocalcium phosphate 1%, premix 1%, choline chloride 0.5%, vitamin C 0.1%, DL-methionine 0.5%, antioxidant 0.1%, and bentonite 10.58%, calculated based on the total weight of the feed.
8. The method for detecting fish adipocyte hyperplasia in vivo according to claim 1, characterized in that: The amount of water added in step S1 is 40% of the total weight of the feed components, and the adding method is small amounts and multiple times in a mixer, and the granulation equipment is a ring die granulator with a diameter of 4 mm.
9. The method for detecting fish adipocyte hyperplasia in vivo according to claim 1, characterized in that: The method of immunofluorescence staining in step S4 comprises the following steps: Step 1. Dewaxing, repairing and sealing of fish fat tissue test samples; Step 2. Perform EdU staining to obtain stained sections; Step 3. Add Caveolin-1 antibody to the stained sections and incubate overnight at 4°C. Step 4. Add FITC fluorescent secondary antibody, incubate at room temperature for 2 hours, then stain the nucleus and seal the slices to prepare fish fat cell detection sections.
10. The method for detecting fish adipocyte hyperplasia in vivo according to claim 1, characterized in that: The image analysis method in step S4 comprises the following steps: Step 1. Observe the fish adipocyte detection slices under a fluorescence microscope and collect images; Step 2. The images were processed and analyzed using ImageJ software.