Method for identifying sheep testicular germ cells based on DDX39A gene immunofluorescence technology

Abstract

The invention discloses a method for identifying sheep testicular germ cells by an immunofluorescence technique based on DDX39A gene, and particularly relates to the technical field of animal breeding, which comprises the following steps: S1, embedding treatment; s2, antigen permeation; s3, antigen repair; s4, closing serum; s5, primary antibody incubation: DDX39A is diluted, and a primary antibody solution is prepared; s6, secondary antibody incubation: selecting a green fluorescence labeled antibody for dilution, and preparing a secondary antibody solution; s7, washing; s8, performing nucleus dyeing; s9, mounting and observing; and S10, analyzing the image. The DDX39A is adopted to replace existing VASA to mark germ cells in the testis, compared with the VASA, the DDX39A gene can participate in multiple processes such as RNA splicing, nuclear output, transcriptional regulation and DNA repair, and the DDX39A gene has the advantages of being diversified in function, suitable for multi-aspect research, high in flexibility and wide in sample source and has wide application prospects clinically.

Description

Technical Field

[0001] The present invention relates to the technical field of animal reproduction, and more specifically to a method for identifying germ cells in sheep testes based on immunofluorescence technology of the DDX39A gene. Background Art

[0002] As the core organ of the male reproductive system, the complex microenvironment of the testis plays an important regulatory role in spermatogenesis and its abnormalities. A variety of cell types in the testis, including germ cells and somatic cells, jointly participate in the process of sperm production and maturation. Precise identification of germ cells is of great significance for understanding reproductive mechanisms, improving reproductive efficiency, and developing new biotechnologies.

[0003] Currently, there are many methods commonly used to identify germ cells in the testis, which can be carried out through various methods such as morphology, immunohistochemistry, immunofluorescence, flow cytometry, molecular biology, functional studies, chromosome analysis, proteomics, epigenetics, and bioinformatics. VASA (DDX4) has been widely recognized as a specific marker for germ cells, and with the help of immunofluorescence technology, germ cells in the testis are labeled red to help people identify germ cells in the testis.

[0004] Although VASA (DDX4) is widely used in germ cell detection, there are still some deficiencies:

[0005] 1. DDX4 is mainly involved in the development of germ cells and has relatively single functions;

[0006] 2. DDX4 is mainly used for germ cell research, and the scope of technical application is relatively limited;

[0007] 3. DDX4 is mainly applicable to samples related to germ cells, resulting in limited sample sources. Summary of the Invention

[0008] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for identifying germ cells in sheep testes based on immunofluorescence technology of the DDX39A gene. By using DDX39A to label germ cells in the testis, this DDX39A gene can participate in multiple processes such as RNA splicing, nuclear export, transcriptional regulation, and DNA repair, and has the advantages of diverse functions, suitability for multi-faceted research, high flexibility, and wide sample sources, so as to solve the problems appearing in the above-mentioned background art.

[0009] To achieve the above object, the present invention provides the following technical solution: A method for identifying germ cells in sheep testes based on immunofluorescence technology of the DDX39A gene, including the following specific steps:

[0010] S1. Embedding treatment: Treat the freshly collected sheep testis tissue and make it into sections;

[0011] S1.1. Tissue washing and fixation: The freshly collected sheep testicular tissue is thoroughly washed 3 times with PBS buffer at certain intervals each time to ensure tissue cleanliness;

[0012] S1.2. Dehydration treatment: After fixation, the tissue is taken out of the fixative and rinsed 3 times with PBS buffer on a shaker for 30 min each time to remove the excess fixative;

[0013] S1.3. Clearing treatment: After dehydration, the tissue is transferred to a mixed solution of ethanol and xylene and soaked for 2 h, then briefly replaced with pure xylene solution and continued to be soaked until the tissue becomes clear;

[0014] S1.4. Impregnation process: After clearing, the tissue is transferred to a solution mixed with xylene and paraffin, gradually transitioned to pure paraffin solution, and ensure that the tissue is completely impregnated with paraffin;

[0015] S1.5. Embedding pretreatment: After impregnation, the tissue is further soaked in clean embedding paraffin for 30 min to ensure complete miscibility of the paraffin inside the tissue with the embedding paraffin;

[0016] S1.6. Tissue embedding: The pretreated tissue is quickly placed into a mold filled with embedding paraffin, and the tissue position is adjusted to ensure that the paraffin cools and solidifies naturally;

[0017] S1.7. Tissue dewaxing: The sections are pre-dried in an oven at 42 °C, and then xylene is used to dissolve the paraffin in the tissue sections;

[0018] S1.8. Tissue rehydration: After the paraffin is completely dissolved, the sections are soaked in 100%, 100%, 90%, 80%, 70% ethanol in sequence, and finally rinsed 3 times with PBS buffer for 3 min each time;

[0019] S2. Antigen permeabilization: The sections are permeabilized with 0.1% TritonX-100 solution and finally rinsed 3 times with PBS buffer for 3 min each time;

[0020] S3. Antigen repair: Use high-pressure repair for 20 min;

[0021] S4. Serum blocking: Use an immunohistochemistry special pen to draw a circle around the tissue, drop serum to cover the tissue, and the sections are placed in a wet box and incubated at room temperature for 1 h;

[0022] S5, Primary antibody incubation: Dilute DDX39A to prepare the primary antibody solution. Take out the sections, shake off the liquid around the tissue, add the primary antibody solution dropwise, and incubate overnight at 4°C in a humidified box. The next day, take out the sections to warm them to room temperature, wash them 3 times with PBS buffer for 2 minutes each time. Then soak and wash them 3 times in PBS for 3 minutes each time.

[0023] S6, Secondary antibody incubation: Select a green fluorescent-labeled antibody for dilution to prepare the secondary antibody solution. Take out the sections, blot the liquid around the tissue dry, and then add 100 μL of the fluorescent-labeled secondary antibody solution and incubate in a humidified box for 1 hour.

[0024] S7, Washing: Wash 3 times with PBS for 3 minutes each time.

[0025] S8, Nuclear staining: Blot the water stains on the glass slide dry, then add 100 μL of DAPI and stain for 10 minutes.

[0026] S9, Mounting and observation: Wash 3 times with PBS, mount the slides, and take pictures under a fluorescence microscope.

[0027] S10, Image analysis.

[0028] In a preferred embodiment, the washed testicular tissue in step S1.1 is cut into small pieces with a thickness of 0.5 cm and placed in a container containing 4% paraformaldehyde for overnight fixation for 14 - 16 hours.

[0029] In a preferred embodiment, the tissue in step S1.2 is dehydrated by a series of ethanol solutions with increasing concentrations starting from 30% to 100%, and the treatment time for each concentration of ethanol is 30 minutes.

[0030] In a preferred embodiment, the entire clearing process in step S1.3 is carried out in a fume hood.

[0031] In a preferred embodiment, the infiltration process in step S1.4 is carried out in a constant-temperature oven at 68°C to keep the paraffin in a liquid state, and the container is shaken every 30 minutes to promote the uniform penetration of the paraffin.

[0032] In a preferred embodiment, after the paraffin solidifies in step S1.6, the embedded tissue block is taken out of the mold and marked according to the previous records to complete the entire embedding process.

[0033] In a preferred embodiment, the mounting operation in step S9 is as follows: Mount the slides with an anti-fluorescence quenching mounting medium and cover with a coverslip.

[0034] In step S9, when observing the sections under a fluorescence microscope, select appropriate excitation and emission wavelengths and take images.

[0035] In a preferred embodiment, in step S10, an image analysis software is used to quantitatively analyze the fluorescence signal and evaluate the distribution and quantity of germ cells.

[0036] Technical effects and advantages of the present invention:

[0037] 1. Aiming at the deficiencies of DDX4 applied in the existing germ cell identification technology, the DDX39A gene adopted by the present invention can participate in multiple processes such as RNA splicing, nuclear export, transcriptional regulation, and DNA repair, and has the advantages of diverse functions and suitability for various aspects of research;

[0038] 2. The DDX39A gene adopted by the present invention has a wide expression and can be used in a variety of experimental techniques (such as RNA metabolism, gene expression regulation, DNA repair, etc.), with high flexibility and a broader clinical application prospect;

[0039] 3. The DDX39A gene adopted by the present invention is also applicable to a variety of tissues and cell types, and the sample sources are extensive. Description of the drawings

[0040] Figure 1 It is a schematic diagram of immunofluorescence staining of VASA in sheep testicular tissue;

[0041] Figure 2 It is a schematic diagram of immunofluorescence staining of DDX39A in sheep testicular tissue;

[0042] Figure 3 It is a schematic diagram of immunofluorescence staining of DDX39A and VASA in sheep testicular tissue. Detailed implementation manners

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0044] The present invention provides a method for identifying sheep testicular germ cells by an immunofluorescence technique based on the DDX39A gene, including the following specific steps:

[0045] 1) Tissue cleaning and fixation: The freshly collected sheep testicular tissue is first thoroughly cleaned three times with PBS buffer, with a certain time interval each time to ensure tissue cleanliness. The cleaned testicular tissue is cut into small pieces (about 0.5 cm thick) and placed in a container containing 4% paraformaldehyde for overnight fixation, usually lasting for 14 to 16 h.

[0046] 2) Dehydration treatment: After fixation, the tissue is taken out of the fixative and rinsed 3 times with PBS buffer on a shaker, 30 minutes each time. After rinsing thoroughly with PBS, the tissue is dehydrated step by step on a shaker using ethanol with concentrations of 30%, 50%, 70%, 80%, 90%, 100% (I) and 100% (II), with each dehydration step lasting 30 minutes.

[0047] 3) Clearing treatment: The dehydrated porcine testis tissue is immediately placed in a mixed solution of ethanol:xylene = 1:1 and soaked for 2 hours. Then it is changed to xylene I and soaked for another 10 minutes, and then changed to xylene II and soaked until most of the tissue becomes transparent, completely removing the fat in the tissue. The clearing is also carried out on a shaker, and because xylene has extremely strong volatility, this step is carried out entirely in a fume hood.

[0048] 4) Impregnation process: After clearing, the tissue is transferred to a mixed solution of xylene:paraffin = 1:1 and soaked for 2 hours. After that, the container is changed, and the tissue is immersed in paraffin I for 1 hour, then changed to paraffin II and soaked for another 2 hours to completely remove the xylene in the tissue. Since it is necessary to ensure that the paraffin remains in a liquid state all the time, this step is carried out in a constant temperature oven at 68°C (the same for the following steps), and the tissue is inverted and shaken every 30 minutes during the impregnation process.

[0049] 5) Embedding pretreatment: After impregnation, the container is changed, and the tissue is placed in clean embedding paraffin and soaked for 30 minutes to ensure that the paraffin in the tissue and the embedding paraffin are fully miscible.

[0050] 6) Tissue embedding: Pour the embedding paraffin into the embedding mold, quickly put in the tissue and adjust the position of the tissue block, make a record, let the paraffin cool naturally, and after the paraffin completely solidifies, take out the embedded tissue from the mold and make corresponding marks according to the record to complete the embedding.

[0051] 7) Tissue dewaxing: The sections are pre-dried in an oven at 42°C in advance, and then xylene is used to dissolve the paraffin in the tissue sections.

[0052] 8) Tissue rehydration: After the paraffin is completely dissolved, the sections are soaked in the order of 100%, 100%, 90%, 80%, 70% ethanol in sequence, and finally rinsed 3 times with PBS buffer, 3 minutes each time.

[0053] 9) Antigen permeabilization: The sections are permeabilized with 0.1% TritonX-100 solution, and finally rinsed 3 times with PBS buffer, 3 minutes each time.

[0054] 10) Antigen repair: Use high-pressure repair for 20 minutes.

[0055] 11) Serum blocking: Use a special immunohistochemistry pen to draw a circle around the tissue and add serum to cover the tissue. Incubate the sections in a wet box at room temperature for 1 h.

[0056] 12) Primary antibody incubation: Dilute DDX39A according to the instruction manual (1:200). Take out the sections, shake off the liquid around the tissue, add the primary antibody solution, and incubate in a wet box at 4 °C overnight. The next day, take out the sections and let them warm to room temperature, then wash them 3 times with PBS buffer for 2 min each time; then soak and wash them in PBS 3 times for 3 min each time.

[0057] 13) Secondary antibody incubation: Select the green fluorescence-labeled antibody - FITC for dilution to prepare the secondary antibody solution. Take out the sections, blot dry the liquid around the tissue, then add 100 μL of the fluorescence-labeled secondary antibody solution and incubate in a wet box for 1 h.

[0058] 14) Washing: Wash 3 times with PBS for 3 min each time.

[0059] 15) Nuclear staining: Blot dry the water stain on the glass slide, then add 100 μL of DAPI and stain for 10 min.

[0060] 16) Mounting and staining: Wash 3 times with PBS, mount the slides, and observe and take pictures under a fluorescence microscope (the excitation / emission wavelength selected during observation is 494 / 518 nm) to obtain the Figure 2 photos as shown. Figure 2 The germ cells labeled by DDX39A appear green.

[0061] 17) Image analysis: Use image analysis software (such as ImageJ) to quantitatively analyze the fluorescence signal and evaluate the distribution and quantity of germ cells.

[0062] Set control example 1, replace the primary antibody solution in step 12) with VASA dilution solution, replace the secondary antibody solution in step 13) with Alexa Fluor 594 dilution solution, and then repeat the remaining steps to obtain the Figure 1 photos as shown. Figure 1 The germ cells labeled by VASA appear red.

[0063] Set control example 2, replace the primary antibody solution in step 12) with VASA dilution solution and DDX39A dilution solution, replace the secondary antibody solution in step 13) with Alexa Fluor 594 dilution solution and FITC dilution solution, and then repeat the remaining steps to obtain the Figure 3 photos as shown. Figure 3 The green cells stained by DDX39A and the red cells stained by VASA show obvious overlapping, indicating that the DDX39A gene can be used to label germ cells, verifying the feasibility of the proposed solution of the present invention.

[0064] Finally, the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for identifying sheep testicular germ cells based on the DDX39A gene by immunofluorescence technology, characterized in that: It includes the following specific steps: S1. Embedding treatment: The freshly collected sheep testis tissue is processed to make sections; S2. Antigen permeabilization: The sections are permeabilized with 0.1% Triton X-100 solution, and then rinsed 3 times with PBS buffer, 3 minutes each time; S3. Antigen repair: Use high-pressure repair for 20 minutes; S4. Serum blocking: Use an immunohistochemistry special pen to draw a circle around the tissue, drop serum to cover the tissue, and incubate the sections in a wet box at room temperature for 1 hour; S5. Primary antibody incubation: Dilute DDX39A to prepare the primary antibody solution. Take out the sections, shake off the liquid around the tissue, drop the primary antibody solution, and incubate in a wet box at 4°C overnight; The next day, take out the sections to restore to room temperature, wash 3 times with PBS buffer, 2 minutes each time; Then soak and wash in PBS 3 times, 3 minutes each time; S6. Secondary antibody incubation: Select a green fluorescent labeled antibody for dilution to prepare the secondary antibody solution. Take out the sections, blot dry the liquid around the tissue, and then drop 100 μL of the fluorescent labeled secondary antibody solution, and incubate in a wet box for 1 hour; S7. Washing: Wash 3 times with PBS, 3 minutes each time; S8. Nuclear staining: Blot dry the water stain on the glass slide, then drop 100 μL of DAPI and stain for 10 minutes; S9. Sealing and observation: Wash 3 times with PBS, seal the slides, and take pictures under a fluorescence microscope; S10. Image analysis.

2. The method for identifying sheep testicular germ cells by immunofluorescence technique based on the DDX39A gene according to claim 1, wherein: The specific steps of the embedding treatment in step S1 are as follows: S1.

1. Tissue cleaning and fixation: The freshly collected sheep testis tissue is thoroughly washed 3 times with PBS buffer; S1.

2. Dehydration treatment: After fixation, the tissue is taken out of the fixative and rinsed 3 times with PBS buffer on a shaker, 30 minutes each time, to remove the excess fixative; S1.

3. Clearing treatment: After dehydration, the tissue is transferred to a mixed solution of ethanol and xylene and soaked for 2 hours, then briefly replaced with pure xylene solution and continued to soak until the tissue becomes transparent; S1.

4. Impregnation process: After clearing, the tissue is transferred to a solution mixed with xylene and paraffin, and gradually transitioned to a pure paraffin solution to ensure that the tissue is completely impregnated with wax; S1.

5. Embedding pretreatment: After impregnation with wax, the tissue is further soaked in clean embedding paraffin for 30 minutes to ensure that the wax inside the tissue is completely miscible with the embedding paraffin; S1.

6. Tissue embedding: The pretreated tissue is quickly placed into a mold filled with embedding paraffin, and the tissue position is adjusted to ensure that the paraffin cools and solidifies naturally to make sections; S1.

7. Tissue dewaxing: The sections are pre-dried in an oven at 42°C, and then xylene is used to dissolve the paraffin in the tissue sections; S1.

8. Tissue rehydration: After the paraffin is completely dissolved, the sections are soaked in 100%, 100%, 90%, 80%, 70% ethanol in sequence and finally rinsed 3 times with PBS buffer, 3 minutes each time.

3. The method for identifying sheep testicular germ cells by immunofluorescence technology based on the DDX39A gene according to claim 2, wherein: The washed testis tissue in step S1.1 is cut into small pieces with a thickness of 0.5 cm and placed in a container containing 4% paraformaldehyde for overnight fixation for 14 - 16 hours.

4. The method for identifying sheep testicular germ cells by immunofluorescence technology based on the DDX39A gene according to claim 2, wherein: The tissue in step S1.2 is dehydrated in a gradient manner through a series of ethanol solutions with increasing concentrations, starting from 30% up to 100%, and the treatment time for each ethanol concentration is 30 min.

5. The method for identifying sheep testicular germ cells based on the immunofluorescence technique of the DDX39A gene according to claim 2, wherein: The entire clearing process in step S1.3 is carried out in a fume hood.

6. The method for identifying sheep testicular germ cells by immunofluorescence technology based on the DDX39A gene according to claim 2, wherein: The infiltration process in step S1.4 is carried out in a constant-temperature oven at 68 °C to keep the paraffin in a liquid state, and the container is shaken every 30 min.

7. The method for identifying sheep testicular germ cells based on the immunofluorescence technique of the DDX39A gene according to claim 2, characterized in that: After the paraffin in step S1.6 solidifies, the embedded tissue block is removed from the mold and labeled according to the previous records, completing the entire embedding process.

8. The method for identifying sheep testicular germ cells by immunofluorescence technology based on the DDX39A gene according to claim 1, characterized in that: The operation of mounting the slides in step S9 is as follows: mount the slides with an anti-fluorescence quenching mounting medium and cover them with a coverslip; When observing the sections using a fluorescence microscope in step S9, select appropriate excitation and emission wavelengths and take images.

9. The method for identifying sheep testicular germ cells by immunofluorescence technology based on the DDX39A gene according to claim 1, characterized in that: In step S10, use image analysis software to quantitatively analyze the fluorescence signals and evaluate the distribution and quantity of germ cells.

Images