Immunofluorescent staining method based on lycoris radiate meiosis cell protein

Abstract

The invention belongs to the technical field of biology, and discloses a lycoris radiata meiosis cell protein immunofluorescence staining method, which comprises the following steps: 1) treating a fixed anther to obtain a pollen mother cell in a meiosis period; 2) performing enzyme treatment to decompose cytoplasm of the meiotic cells; (3) carrying out slide preparation and fluorescent staining by using the treated meiotic cells; and 4) observing the positioning signal of the target protein under a fluorescence microscope. The pollen mother cells are treated by using glucanase and the like, so that a thicker callose protective layer on the outer layer of the lycoris radiate pollen mother cells is effectively decomposed, and the binding of an antibody and chromosomes is facilitated; meanwhile, a film preparation method is improved, and expression of fluorescence signals is effectively enhanced. The method is of great significance in researching subcellular localization of proteins with different functions in pollen mother cells of lycoris radiate in a meiosis period by using an immunofluorescence localization technology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method based on immunofluorescence staining of meiotic cell proteins of Lycoris radiata. Background Art

[0002] Lycoris genus is in the meiotic process, and protein expression and location in pollen mother cell have unique biological significance.Existing immunofluorescence staining method has some limitations when processing the meiotic cells of this type of bulbous plants of Lycoris radiata, such as problems such as cell wall thickness, poor antibody permeability, weak fluorescent signal, etc., which have affected the accurate research on the subcellular localization of proteins with different functions in Lycoris radiata meiotic cells.In the prior art, for example, the patent applications of publication numbers CN102559909A and CN101074952A all use cellulase and pectinase to carry out enzymolysis, but are not suitable for this type of bulbous plants of Lycoris radiata.Therefore, developing a kind of high-efficiency protein immunofluorescence staining method for Lycoris radiata meiotic cells has important scientific value and application prospect. Summary of the Invention

[0003] The purpose of the present invention is to provide a high-efficiency protein immunofluorescence staining method for Lycoris radiata meiotic cells, so as to solve the deficiencies in the prior art and improve the staining effect and detection accuracy.

[0004] In one aspect, the present invention provides a method for immunofluorescence staining of meiotic cell proteins based on Lycoris radiata, comprising the following steps: S1. Obtaining Lycoris radiata meiotic cells: Fixing Lycoris radiata flower buds in the meiotic stage, separating anthers and squeezing out pollen mother cells; S2, two-stage enzymatic treatment: The first stage: the pollen mother cells are immersed in glucanase solution and enzymatically hydrolyzed; The second stage: after cleaning, soaking in mixed enzyme solution for enzymatic hydrolysis; The enzymes in the mixed enzyme solution include cellular helicase, cellulase and pectinase; S3, cell smear fixation after enzymatic hydrolysis; S4, incubate primary antibody and fluorescently labeled secondary antibody sequentially; S5. Observe under a fluorescence microscope after sealing.

[0005] Preferably, the Lycoris radiata is selected from Lycoris radiata, Lycoris radiata or Lycoris radiata (also known as yellow flower Lycoris radiata).

[0006] Preferably, in step S1, the fixative for fixing Lycoris radiata flower buds during the meiotic division period is Carnoy's fixative; the Carnoy's fixative comprises anhydrous ethanol and acetic acid in a volume ratio of 3:1; the fixation time is ≥24 hours to maintain the cell morphology and structure. The meiotic division period is the first meiotic division period or the second meiotic division period.

[0007] Preferably, in step S2, the mass concentration of the glucanase solution is 2.5%; The mass concentration of the mixed enzyme solution is 0.3%, and it is dissolved in 10mM citric acid buffer at pH 4.5, wherein the ratio of cellular helicase, cellulase and pectinase is 1:1:1; The soaking time for enzymatic hydrolysis using glucanase solution shall not be less than 1 hour, and the soaking time for enzymatic hydrolysis using mixed enzyme solution shall not be less than 4 hours.

[0008] The two-stage enzymatic hydrolysis can effectively decompose the callose protective layer on the outer layer of Lycoris radiata pollen mother cells, improve the permeability of antibodies, and enhance the expression of fluorescence signals.

[0009] The main component of the callose protective layer on the outer layer of Lycoris radiata pollen mother cells is β-1,3-glucan, a densely structured polysaccharide that hinders antibodies from entering the cell and binding to the target protein.

[0010] The glucanase (mass concentration 2.5%) used in the first stage can specifically hydrolyze β-1,3-glycosidic bonds. Its function is to decompose the callose protective layer to initially reduce the physical barrier effect of the outer layer of the cell, providing an "entrance" for subsequent enzymatic hydrolysis and antibody penetration.

[0011] If glucanase is used alone, it can only partially break down callus, but cannot process the cell wall of the pollen mother cell itself (containing cellulose, pectin, etc.) and the intercellular connecting substances. The cell still maintains a relatively complete outer layer structure, the antibody penetration rate is low, and the fluorescence signal is weak.

[0012] Therefore, the enzyme mixture used in the second stage consists of cellular helicase, cellulase, and pectinase in a 1:1:1 ratio (mass concentration 0.3%, dissolved in 10mM citric acid buffer at pH 4.5). Its functions are: Cellulase: breaks down cellulose in the cell wall (the main component of plant cell walls), destroying the rigid structure of the cell; Pectinase: degrades pectin substances between cells, promotes cell dispersion, and avoids uneven staining caused by cell aggregation; Cell helicase: helps loosen the protein-polysaccharide complexes within the cell, further reducing the material barrier inside the cell.

[0013] If the mixed enzyme is used alone: ​​Since the thick callose protective layer of Lycoris radiata has not been destroyed in advance, the mixed enzyme has difficulty in contacting the cell wall and intercellular components, the enzymatic hydrolysis efficiency is low, the antibody cannot enter, and there is almost no effective fluorescent signal.

[0014] The synergistic effect of the two-step enzymatic hydrolysis not only solves the problem of incomplete decomposition of complex structures by a single enzyme, but also avoids cell morphological damage caused by excessive enzymatic hydrolysis by controlling the enzymatic hydrolysis time (1.5h for glucanase + 5h for mixed enzyme). Ultimately, the antibody achieves efficient penetration and full binding to the target protein, significantly enhancing the intensity and stability of the fluorescence signal.

[0015] In the embodiment of the present invention, it is preferred that the soaking time for enzymolysis using the glucanase solution is 1.5 hours, and the soaking time for enzymolysis using the mixed enzyme solution is 5 hours.

[0016] Furthermore, in step S3, when the cell smear after enzymatic hydrolysis is fixed, water is first added to the enzymatically hydrolyzed cells and crushed (the cell chromosomes are still clumped together after enzymatic hydrolysis and need to be moderately crushed to unfold them), and then 10-20 μL of 60% acetic acid solution is added to mix the smear, and the position is marked after drying and fixing.

[0017] The specific steps are as follows: S201, placing the pollen mother cells obtained in step S1 and the remaining broken anther tissues into a centrifuge tube, adding 2.5% glucanase and soaking for enzymatic hydrolysis for not less than 1 hour; S202, adding an appropriate amount of double deionized water (ddH2O) to the centrifuge tube after the enzymatic hydrolysis in step S201, washing the enzyme solution, centrifuging and removing the supernatant; S203, adding 0.3% mixed enzyme to the centrifuge tube after removing the supernatant in step S202, and soaking for enzymatic hydrolysis for not less than 4 hours; S204, adding an appropriate amount of ddH2O to the centrifuge tube after the enzymatic hydrolysis in step S203, washing the enzyme solution, centrifuging and removing the supernatant; S205. Transfer the remaining precipitate in the centrifuge tube from which the supernatant was removed in step S204 and place it on a glass slide; add ddH2O and grind until it becomes a paste; add 10-20 μL of 60% acetic acid solution and mix well, then smear the slide. Subsequently, place the slide on a 45°C slide dryer for fixation. Then, rinse the sample with 1 mL of Carnoy's fixative and mark the smear position of the sample.

[0018] Preferably, before incubation in step S4, the fixed sample is further subjected to microwave treatment with a citrate solution, followed by soaking for 3-5 minutes; Microwave treatment of fixed samples with citrate solution has two effects: 1. Antigen Retrieval In paraffin sections or formaldehyde-fixed tissues, proteins form chemical bonds due to cross-linking, masking antigen epitopes and causing the immunofluorescence staining signal to weaken or disappear.

[0019] Citrate buffer (usually pH 6.0) is heated (e.g., in a microwave oven, pressure cooker, or water bath) to break cross-linked protein peptide chains, re-exposing the antigen epitope and restoring its ability to bind to the antibody.

[0020] Significantly improve staining signal intensity, reduce nonspecific background, and lower false negative rate.

[0021] 2. Maintain pH stability Citrate buffer is a weakly acidic buffer system (pH 3-6) that can resist pH fluctuations during heating, ensuring the stability of the antigen retrieval environment and avoiding the impact of pH changes on the antigen epitope structure.

[0022] After soaking, the sample was soaked in buffer 1 for 1 hour in the dark; the buffer 1 was prepared by dissolving 1 mL of Triton X-100 in 100 mL of 10×PBS buffer and adding water to make the volume to 1 L, with a pH of 7.4.

[0023] Furthermore, a 10 mM citrate solution (specifically, a sodium citrate solution is used in the embodiment of the present invention) is used, and the power during microwave treatment is 50 p, and the microwave treatment is heated until the solution is close to boiling, that is, 90-100°C.

[0024] Preferably, in step S4, the primary antibody is a ready-to-use goat serum primary antibody, and the antibody is incubated at 4° C. in the dark for ≥48 h; The fluorescently labeled secondary antibody is a ready-to-use goat serum fluorescently labeled secondary antibody, and is incubated at 37°C in the dark for ≥70 min. During the incubation process, the temperature and time are controlled to ensure sufficient binding of the antibody to the target protein.

[0025] After incubation with the primary antibody, the cells were washed with buffer 2; the buffer 2 was prepared by dissolving 1 mL of Tween-20 in 100 mL of 10× PBS buffer and adding water to make the volume 1 L, with a pH of 7.4.

[0026] The specific steps include: S301. Slowly rinse the sample obtained in steps S2 and S3 with 1 mL of ice-cold Carnoy's fixative, air-dry, transfer to 10 mM citrate solution, and soak in it for 3-5 minutes after heating in a microwave oven. S302, quickly take out the sample after being treated in step S301, and soak it in buffer 1 in the dark for 1 hour; S303, taking out the sample after the treatment in step S302 and placing it in a moisturizing box, adding 40 μL of ready-to-use goat serum to the sample to block nonspecific binding, covering the sample and incubating it in the dark at room temperature for 40 minutes; S304, removing the cover glass of the sample after treatment in step S303, adding 40 μL of ready-to-use goat serum primary antibody on one side, and incubating at 4°C in the dark for 48 hours after covering the sample; S305, remove the cover glass of the sample after the treatment in step S304, wash it three times with buffer 2, each time for 15 minutes, and preheat the moisturizing box at 37°C; S306. Take out the sample after treatment in step S305 and place it in a preheated moisturizing box. Add 40 μL of ready-to-use goat serum secondary antibody to one side and incubate at 37°C in the dark for 70 min. S307. Remove the coverslip of the sample after treatment in step S306, wash with buffer 2 three times, 20 minutes each time, add 8 μL of DAPI (i.e., 4,6-diamidino-2-phenylindole, 5 μg / mL) in the center of the sample, and cover the sample.

[0027] The beneficial effects of the present invention are: (1) Through optimized enzymatic hydrolysis, the callose protective layer on the outer layer of Lycoris radiata pollen mother cells was effectively decomposed, the permeability of the antibody was improved, and the expression of the fluorescence signal was enhanced.

[0028] (2) Improved preparation and staining methods can better preserve the morphology and structure of cells and improve the stability and repeatability of staining effects.

[0029] (3) It is suitable for a variety of Lycoris plants, such as seaside Lycoris, spring Lycoris and Lycoris radiata, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the fluorescence microscopy observation result of immunofluorescence staining of the localized homologous recombination protein HEI10 in meiotic cells of Lycoris radiata in Example 1 of the present invention. Scale bar = 10 μm.

[0031] Figure 2 This is the fluorescence microscopy observation result of immunofluorescence staining of the localized homologous recombination protein HEI10 in Hudixiao meiotic cells in Example 2 of the present invention. Scale bar = 10 μm.

[0032] Figure 3 The figure shows the comparison of immunofluorescence staining of SYN1 and HEI10 in meiotic stage cells of Lycoris radiata before the improvement of the immunofluorescence protein staining technique of the present invention. Scale bar = 10 μm.

[0033] Figure 4The figure shows the comparison of immunofluorescence staining of SYN1 and HEI10 in meiotic stage cells of Lycoris radiata after the improvement of the immunofluorescence protein staining technique of the present invention. Scale bar = 10 μm. DETAILED DESCRIPTION

[0034] Example 1: Immunofluorescence staining of the homologous recombination protein HEI10 in Lycoris radiata meiotic cells 1. Take the flower buds of Lycoris radiata in the meiotic period and immerse them in Carnoy's fixative (anhydrous ethanol and acetic acid prepared in a volume ratio of 3:1) for more than 24 hours. Take out the anthers and place them in Carnoy's fixative until they are decolorized.

[0035] 2. Remove the anthers and wash them twice with ddH2O, then transfer them to 10 mM sodium citrate solution and wash once.

[0036] 3. Use a syringe to cut part of the anther and place it on a glass slide to squeeze and squeeze out the pollen mother cells.

[0037] 4. Put the pollen mother cells and broken anther tissue into a centrifuge tube, add 2.5% glucanase and soak for enzymatic hydrolysis for 1.5 hours.

[0038] 5. Add appropriate amount of ddH2O to the centrifuge tube to wash the enzyme solution, centrifuge and remove the supernatant.

[0039] 6. Add 0.3% mixed enzyme (0.3% (w / v) cell helicase, 0.3% (w / v) cellulase, and 0.3% (w / v) pectinase, dissolved in 10 mM citric acid buffer, pH 4.5) and soak for enzymatic hydrolysis for 5 h.

[0040] 7. Add appropriate amount of ddH2O again to wash the enzyme solution, centrifuge and remove the supernatant.

[0041] 8. Transfer the remaining precipitate to a glass slide, add ddH2O and grind until it becomes a paste, add 10-20μL of 60% acetic acid solution, mix well, smear, dry and fix, and mark the smear position of the sample.

[0042] 9. Rinse the sample slowly with 1 mL of ice-cold Carnoy's fixative, air-dry it, transfer it to 10 mM sodium citrate solution and soak it in a microwave oven (power p = 50, the solution is close to boiling, the temperature range is 90-100 ° C), and soak it for 4 minutes.

[0043] 10. Quickly remove the sample and soak it in buffer 1 (1 mL Triton X-100 dissolved in 100 mL 10× PBS buffer, pH = 7.4, and dilute to 1 L with deionized water. 10× PBS buffer (pH = 7.4) is prepared by weighing 80 g sodium chloride, 2 g potassium chloride, 35.814 g disodium hydrogen phosphate, and 2.45 g potassium dihydrogen phosphate, then adding deionized water to dilute to 1 L and adjust the pH to 7.4.) in the dark for 1 h.

[0044] 11. Place the sample in a moisturizing box, add 40 μL of ready-to-use goat serum to block nonspecific binding, cover the slide and incubate at room temperature in the dark for 40 minutes.

[0045] 12. Add 40 μL of ready-to-use goat serum primary antibody, cover the slide and incubate at 4°C in the dark for 48 minutes.

[0046] 13. Wash three times with buffer 2 (1 mL Tween-20 dissolved in 100 mL 10× PBS buffer, pH 7.4, and dilute to 1 L with deionized water), each time for 15 minutes, and preheat the moisturizing box at 37°C.

[0047] 14. Place the sample in a preheated moisturizing box, add 40 μL of ready-to-use goat serum secondary antibody, and incubate at 37°C in the dark for 70 minutes.

[0048] 15. Wash three times with buffer 2, 20 min each time, add 8 μL of DAPI (5 μg / mL) to the center of the sample, and cover the slide.

[0049] 16. Apply nail polish evenly around the coverslip, air dry it, and store it at -20°C away from light.

[0050] 17. Use a fluorescence microscope to observe and record the localization signal of the target protein (HEI10 signal).

[0051] The HEI10 protein is a key protein that catalyzes type I homologous crossover formation. In Arabidopsis, HEI10 signals are detected on chromosomes during the leptotene stage, where numerous and small HEI10 foci appear. Larger, brighter HEI10 foci begin to appear during the zygotene and pachytene stages. As meiosis progresses, the small HEI10 foci on the chromosomes gradually converge into larger, brighter HEI10 foci, ultimately forming approximately 10 HEI10 signals by diakinesis.

[0052] like Figure 1As shown, numerous HEI10 signals, including a few large and bright HEI10 signals, were present on the chromosomes of Lycoris radiata during the pachytene stage. However, the HEI10 signal decreased significantly during diakinesis, with only large, bright HEI10 signals observed on the chromosomes. This result is similar to that observed in other species, demonstrating the reliability of this experimental method. Furthermore, we observed at least one HEI10 signal on each pair of homologous chromosomes during diakinesis, indicating that at least one type I chiasmata formed between each pair of homologous chromosomes in Lycoris radiata, ensuring at least one genetic exchange between homologous chromosomes and normal segregation of homologous chromosomes.

[0053] Example 2: Immunofluorescence staining of the localization of the homologous recombination protein HEI10 in meiotic cells of Hudixiao Take the flower buds of Rhizoma Coptidis in the meiotic period, and the remaining steps are the same as those in Example 1.

[0054] Fluorescence microscopy results Figure 2 As shown, in Lycoris radiata, HEI10 signals appear specifically on chromosomes, and their localization pattern is similar to that in Lycoris radiata. Numerous HEI10 foci were observed during the pachytene stage, which gradually condensed and fused into large HEI10 foci.

[0055] Comparative Example 1: Immunofluorescence staining of SYN1 and HEI10 signals in meiotic cells of Lycoris radiata before improvement 1. Take the flower buds of Lycoris radiata in the meiotic period and immerse them in Carnoy's fixative (anhydrous ethanol and acetic acid prepared in a volume ratio of 3:1) for more than 24 hours. Take out the anthers and place them in Carnoy's fixative until they are decolorized.

[0056] 2. Remove the anthers and wash them twice with ddH2O, then transfer them to 10 mM sodium citrate solution and wash once.

[0057] 3. Place the tube in a centrifuge, add 0.3% mixed enzyme (0.3% (w / v) cell helicase, 0.3% (w / v) cellulase, and 0.3% (w / v) pectinase, dissolved in 10 mM citric acid buffer, pH 4.5), and soak for enzymatic hydrolysis for 3 hours.

[0058] 4. Add appropriate amount of ddH2O to clean the enzyme solution, transfer the enzymatically hydrolyzed anthers to a glass slide, add ddH2O and grind until it becomes a paste, add 10-20μL of 60% acetic acid solution, mix well, smear, dry and fix, and mark the smear position of the sample.

[0059] 5. Rinse the sample slowly with 1 mL of ice-cold Carnoy's fixative, air-dry it, transfer it to 10 mM sodium citrate solution and soak it in a microwave oven (power p = 50, the solution is close to boiling, the temperature range is 90-100 ° C), and soak it for 4 minutes.

[0060] 6. Quickly remove the sample and soak it in buffer 1 (1 mL Triton X-100 dissolved in 100 mL 10× PBS buffer, pH 7.4, and dilute to 1 L with deionized water. 10× PBS buffer (pH 7.4) is prepared by weighing 80 g sodium chloride, 2 g potassium chloride, 35.814 g disodium hydrogen phosphate, and 2.45 g potassium dihydrogen phosphate, then dilute to 1 L with deionized water and adjust the pH to 7.4.) in the dark for 40 min.

[0061] 7. Place the sample in a moisturizing box, add 40 μL of ready-to-use goat serum to block nonspecific binding, cover the slide and incubate at room temperature in the dark for 40 minutes.

[0062] 8. Add 40 μL of ready-to-use goat serum primary antibody, cover the slide and incubate at 4°C in the dark for 48 h.

[0063] 9. Wash three times with buffer 2 (1 mL Tween-20 dissolved in 100 mL 10× PBS buffer, pH 7.4, and dilute to 1 L with deionized water), each time for 15 minutes, and preheat the moisturizing box at 37°C.

[0064] 10. Place the sample in a preheated moisturizing box, add 40 μL of ready-to-use goat serum secondary antibody, and incubate at 37°C in the dark for 70 minutes.

[0065] 11. Wash three times with buffer 2, 15 min each time, add 8 μL of DAPI (5 μg / mL) to the center of the sample, and cover the slide.

[0066] 12. Apply nail polish evenly around the coverslip, air dry, and store in the dark at -20°C.

[0067] 13. Use a fluorescence microscope to observe and record the localization signals of the target protein (SYN1 signal and HEI10 signal).

[0068] Fluorescence microscopy results Figure 3 As shown, SYN1 and HEI10 signals failed to form specific fluorescent signal foci on chromosomes in meiotic cells of Lycoris radiata.

[0069] Example 3: Effect of improved immunofluorescence staining technique for SYN1 and HEI10 signals in meiotic cells of Lycoris radiata 1. Take the flower buds of Lycoris radiata in the meiotic period and immerse them in Carnoy's fixative (anhydrous ethanol and acetic acid prepared in a volume ratio of 3:1) for more than 24 hours. Take out the anthers and place them in Carnoy's fixative until they are decolorized.

[0070] 2. Remove the anthers and wash them twice with ddH2O, then transfer them to 10 mM sodium citrate solution and wash once.

[0071] 3. Use a syringe to cut part of the anther and place it on a glass slide to squeeze out the pollen mother cells (this adds an extra step to the anther processing).

[0072] 4. Put the pollen mother cells and broken anther tissue into a centrifuge tube, add 2.5% glucanase and soak for enzymatic hydrolysis for 1.5 hours (add glucanase treatment step).

[0073] 5. Add appropriate amount of ddH2O to the centrifuge tube to wash the enzyme solution, centrifuge and remove the supernatant.

[0074] 6. Add 0.3% mixed enzyme (0.3% (w / v) cell helicase, 0.3% (w / v) cellulase, and 0.3% (w / v) pectinase, dissolved in 10 mM citric acid buffer, pH 4.5) and soak for enzymatic hydrolysis for 5 h (extend the enzymatic hydrolysis time of the mixed enzyme).

[0075] 7. Add appropriate amount of ddH2O again to wash the enzyme solution, centrifuge and remove the supernatant.

[0076] 8. Transfer the remaining precipitate to a glass slide, add ddH2O and grind until it becomes a paste, add 10-20μL of 60% acetic acid solution, mix well, smear, dry and fix, and mark the smear position of the sample.

[0077] 9. Rinse the sample slowly with 1 mL of ice-cold Carnoy's fixative solution, air-dry it, transfer it to 10 mM sodium citrate solution and soak it in a microwave oven (power p = 50, the solution is close to boiling, the temperature range is 90-100 ° C), and soak it for 4 minutes.

[0078] 10. Quickly remove the sample and soak it in buffer 1 (1 mL Triton X-100 dissolved in 100 mL 10× PBS buffer, pH 7.4, and dilute to 1 L with deionized water. 10× PBS buffer (pH 7.4) is prepared by weighing 80 g sodium chloride, 2 g potassium chloride, 35.814 g disodium hydrogen phosphate, and 2.45 g potassium dihydrogen phosphate, then adding deionized water to dilute to 1 L and adjust the pH to 7.4.) in the dark for 1 h (extend the buffer soaking time).

[0079] 11. Place the sample in a moisturizing box, add 40 μL of ready-to-use goat serum to block nonspecific binding, cover the slide and incubate at room temperature in the dark for 40 minutes.

[0080] 12. Add 40 μL of ready-to-use goat serum primary antibody, cover the slide and incubate at 4°C in the dark for 48 h.

[0081] 13. Wash three times with buffer 2 (1 mL Tween-20 dissolved in 100 mL 10× PBS buffer, pH 7.4, and dilute to 1 L with deionized water), each time for 15 minutes, and preheat the moisturizing box at 37°C.

[0082] 14. Place the sample in a preheated moisturizing box, add 40 μL of ready-to-use goat serum secondary antibody, and incubate at 37°C in the dark for 70 minutes.

[0083] 15. Wash three times with buffer 2 for 20 min each time, add 8 μL of DAPI (5 μg / mL) to the center of the sample, and cover the slide (extend the secondary antibody washing time to further remove non-specific fluorescent signals).

[0084] 16. Apply nail polish evenly around the coverslip, air dry it, and store it at -20°C away from light.

[0085] 17. Use a fluorescence microscope to observe and record the localization signals of the target protein (SYN1 signal and HEI10 signal).

[0086] Fluorescence microscopy results Figure 4 As shown, the antibodies bind well to chromosomes, and SYN1 and HEI10 signals form specific fluorescent signal foci on the chromosomes in meiotic cells of Lycoris radiata.

Claims

1. A method based on immunofluorescence staining of Lycoris radiata meiotic cell proteins, characterized in that: The following steps are involved: S1. Obtaining Lycoris radiata meiotic cells: Fixing Lycoris radiata flower buds in the meiotic stage, separating anthers and squeezing out pollen mother cells; S2, two-stage enzymatic treatment: The first stage: the pollen mother cells are immersed in glucanase solution and enzymatically hydrolyzed; The second stage: after cleaning, soaking in mixed enzyme solution for enzymatic hydrolysis; The enzymes in the mixed enzyme solution include cellular helicase, cellulase and pectinase; S3, cell smear fixation after enzymatic hydrolysis; S4, incubate primary antibody and fluorescently labeled secondary antibody sequentially; S5. Observe under a fluorescence microscope after sealing.

2. The method according to claim 1, wherein the immunofluorescence staining method comprises: The Lycoris radiata is selected from Lycoris radiata, Lycoris radiata or Lycoris radiata.

3. The method according to claim 1, wherein the immunofluorescence staining method comprises: In step S1, the fixative for fixing the flower buds of Lycoris radiata during meiosis is Carnoy's fixative; the Carnoy's fixative comprises anhydrous ethanol and acetic acid in a volume ratio of 3:1; and the fixation time is ≥24 hours; The meiotic division period is the first meiotic division period or the second meiotic division period.

4. The method according to claim 1, wherein the immunofluorescence staining method comprises: In step S2, the mass concentration of the glucanase solution is 2.5%; The mass concentration of the mixed enzyme solution is 0.3%, and it is dissolved in 10 mM citric acid buffer with a pH of 4.5, wherein the ratio of cellular helicase, cellulase and pectinase is 1:1:1; The soaking time for enzymatic hydrolysis using glucanase solution shall not be less than 1 hour, and the soaking time for enzymatic hydrolysis using mixed enzyme solution shall not be less than 4 hours.

5. The method according to claim 4, wherein the immunofluorescence staining method comprises: The soaking time for enzymolysis using glucanase solution was 1.5 h, and the soaking time for enzymolysis using mixed enzyme solution was 5 h.

6. The method according to claim 1, wherein the immunofluorescence staining method comprises: In step S3, when the cell smear after enzymatic hydrolysis is fixed, water is first added to the enzymatically hydrolyzed cells and crushed, and then 10-20 μL of 60% acetic acid solution is added to mix the smear, and the position is marked after drying and fixing.

7. The method according to claim 1, wherein the immunofluorescence staining method comprises: Before incubation in step S4, the fixed sample is further treated with a citrate buffer solution by microwave and then soaked for 3-5 minutes; After soaking, the sample was soaked in buffer 1 for 1 hour in the dark; the buffer 1 was prepared by dissolving 1 mL of Triton X-100 in 100 mL of 10×PBS buffer and adding water to make the volume to 1 L, with a pH of 7.

4.

8. The method according to claim 7, wherein the immunofluorescence staining method comprises: Use 10 mM citrate solution, microwave at 50 pF, and heat to 90-100°C.

9. The method according to claim 1, wherein the immunofluorescence staining method comprises: In step S4, the primary antibody is a ready-to-use goat serum primary antibody, and the mixture is incubated at 4° C. in the dark for ≥48 h; The fluorescently labeled secondary antibody is a ready-to-use goat serum fluorescently labeled secondary antibody, which is incubated at 37° C. in the dark for ≥70 min.

10. The method based on immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 9, characterized in that: After incubation with the primary antibody, the cells were washed with buffer 2; the buffer 2 was prepared by dissolving 1 mL of Tween-20 in 100 mL of 10× PBS buffer and adding water to make the volume 1 L, with a pH of 7.4.

Images