A method for immunofluorescence staining of proteins from Lycoris radiata meiotic cells

Abstract

This invention belongs to the field of biotechnology and discloses a method for immunofluorescence staining of proteins in Lycoris radiata meiotic cells. The method includes the following steps: 1) treating fixed anthers to obtain pollen mother cells in the meiotic stage; 2) treating with enzymes to decompose the cytoplasm of the meiotic cells; 3) preparing slides and performing fluorescent staining on the treated meiotic cells; 4) observing the localization signal of the target protein under a fluorescence microscope. This invention uses dextranase and other enzymes to treat pollen mother cells, effectively decomposing the thick outer callosity protective layer of Lycoris radiata pollen mother cells, which is beneficial for antibody binding to chromosomes; at the same time, it improves the slide preparation method, effectively enhancing the expression of fluorescence signals. This method is of great significance for studying the subcellular localization of proteins with different functions in Lycoris radiata pollen mother cells in the meiotic stage using immunofluorescence localization technology.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for immunofluorescence staining of proteins from Lycoris radiata meiotic cells. Background Technology

[0002] During meiosis in Lycoris radiata plants, protein expression and localization in pollen mother cells have unique biological significance. Existing immunofluorescence staining methods have limitations when processing meiotic cells of bulbous plants like Lycoris radiata, such as thick cell walls, poor antibody permeability, and weak fluorescence signals, affecting the accurate study of the subcellular localization of proteins with different functions in Lycoris radiata meiotic cells. Existing technologies, such as patent applications with publication numbers CN102559909A and CN101074952A, both use enzymatic digestion with cellulase and pectinase, but these are not applicable to bulbous plants like Lycoris radiata. Therefore, developing a highly efficient immunofluorescence staining method for proteins in Lycoris radiata meiotic cells has significant scientific value and application prospects. Summary of the Invention

[0003] The purpose of this invention is to provide a highly efficient protein immunofluorescence staining method for Lycoris radiata meiotic cells, in order to overcome the shortcomings of the prior art and improve the staining effect and detection accuracy.

[0004] On one hand, the present invention provides a method for immunofluorescence staining of proteins from Lycoris radiata meiotic cells, comprising the following steps:

[0005] S1. Obtaining Lycoris radiata meiotic cells: fix the flower buds of Lycoris radiata during meiosis, separate the anthers and squeeze out the pollen mother cells;

[0006] S2, Two-stage enzymatic hydrolysis treatment:

[0007] First stage: Soak pollen mother cells in dextranase solution for enzymatic hydrolysis;

[0008] Second stage: After cleaning, soak in mixed enzyme solution for enzymatic hydrolysis;

[0009] The enzymes in the mixed enzyme solution include cell helicase, cellulase and pectinase;

[0010] S3. Fixation of cell smears after enzymatic digestion;

[0011] S4. Incubate with primary antibody and fluorescently labeled secondary antibody sequentially;

[0012] S5. After mounting, observe under a fluorescence microscope.

[0013] Preferably, the Lycoris radiata is selected from Lycoris radiata, Lycoris radiata, or Lycoris radiata (also known as Lycoris radiata).

[0014] Preferably, in step S1, the fixative for fixing the flower buds of Lycoris radiata during meiosis is Carnos' fixative; the Carnos' fixative is prepared by mixing anhydrous ethanol and acetic acid in a volume ratio of 3:1; the fixation time is ≥24 hours to maintain the morphology and structure of the cells. The meiotic period is either the first meiotic division or the second meiotic division.

[0015] Preferably, in step S2, the mass concentration of the dextranase solution is 2.5%;

[0016] The mixed enzyme solution has a mass concentration of 0.3% and is dissolved in 10mM citrate buffer at pH 4.5, wherein the ratio of cell helicase, cellulase and pectinase is 1:1:1.

[0017] The soaking and enzymatic hydrolysis time with dextranase solution should not be less than 1 hour, and the soaking and enzymatic hydrolysis time with mixed enzyme solution should not be less than 4 hours.

[0018] Two-stage enzymatic hydrolysis can effectively break down the callosity protective layer on the outer layer of Lycoris pollen mother cells, improve antibody permeability, and enhance the expression of fluorescent signals.

[0019] The main component of the callosity protective layer on the outer layer of Lycoris pollen mother cells is β-1,3-glucan, a dense polysaccharide that can prevent antibodies from entering the cell and binding to target proteins.

[0020] The first stage uses a dextranase (2.5% by mass) that specifically hydrolyzes β-1,3-glycosidic bonds. Its function is to break down the callosity protective layer, initially reducing the physical barrier effect of the extracellular layer and providing an "entry point" for subsequent enzymatic digestion and antibody penetration.

[0021] If glucanase is used alone, it can only partially break down callose, but it cannot process the cell wall (containing cellulose, pectin, etc.) and intercellular junctions of the pollen mother cell itself. The cell still maintains a relatively intact outer structure, resulting in low antibody penetration and weak fluorescence signal.

[0022] Therefore, the mixed enzyme used in the second stage consists of cell helicase, cellulase, and pectinase in a 1:1:1 ratio (0.3% by mass, dissolved in 10mM citrate buffer at pH 4.5), and its function is:

[0023] Cellulase: breaks down cellulose (the main component of plant cell walls) in cell walls, thus disrupting the rigid structure of cells;

[0024] Pectinase: Degrades pectin-like substances between cells, promotes cell dispersion, and avoids uneven staining caused by cell aggregation;

[0025] Cellular helicases: help to loosen protein-polysaccharide complexes within cells, further reducing the material barrier inside the cell.

[0026] If the mixed enzyme is used alone: ​​because the thick callus protective layer of Lycoris radiata is not destroyed beforehand, the mixed enzyme has difficulty contacting the cell wall and intercellular components, resulting in low enzymatic hydrolysis efficiency, inability of antibodies to enter, and almost no effective fluorescent signal.

[0027] The two-step enzymatic hydrolysis synergistic effect not only solves the problem of incomplete decomposition of complex structures by a single enzyme, but also avoids cell morphology damage caused by excessive enzymatic hydrolysis by controlling the hydrolysis time (1.5h for glucanase + 5h for mixed enzymes). Ultimately, it achieves efficient antibody penetration and full binding to the target protein, significantly enhancing the intensity and stability of the fluorescence signal.

[0028] In the embodiments of the present invention, the preferred soaking and enzymatic hydrolysis time using dextranase solution is 1.5 hours, and the soaking and enzymatic hydrolysis time using mixed enzyme solution is 5 hours.

[0029] Furthermore, in step S3, when fixing the cell smear after enzymatic digestion, water is first added to the digested cells and they are rolled up (the cell chromosomes are still clumped together after enzymatic digestion, so they need to be rolled up appropriately to spread them out). Then, 10-20 μL of 60% acetic acid solution is added, the smear is mixed, dried, fixed, and the position is marked.

[0030] The specific steps are as follows:

[0031] S201. Place the pollen mother cells obtained in step S1 and the remaining broken anther tissues into a centrifuge tube, add 2.5% dextranase and soak for enzymatic hydrolysis for no less than 1 hour.

[0032] S202. Add an appropriate amount of double-distilled water (ddH2O) to the centrifuge tube after the enzymatic hydrolysis in step S201, wash the enzyme solution, centrifuge and remove the supernatant.

[0033] S203. Add 0.3% mixed enzyme to the centrifuge tube after removing the supernatant in step S202 and soak for enzymatic hydrolysis for no less than 4 hours;

[0034] S204. Add an appropriate amount of ddH2O to the centrifuge tube after the enzymatic hydrolysis in step S203, wash the enzyme solution, centrifuge and remove the supernatant.

[0035] S205. Transfer the remaining precipitate from the centrifuge tube after removing the supernatant in step S204 to a glass slide; add ddH2O and grind into a paste; add 10-20 μL of 60% acetic acid solution, mix well, and smear. Then, place the glass slide on a 45℃ slide dryer for drying and fixation. Next, rinse the sample with 1 mL of Carnoy's fixative and mark the positions of the sample smear.

[0036] Preferably, step S4 includes micro-treating the fixed sample with citrate solution before incubation, followed by soaking for 3-5 minutes.

[0037] Microwave treatment of the fixed sample with citrate solution has two effects:

[0038] 1. Antigen retrieval

[0039] In paraffin sections or formaldehyde-fixed tissues, proteins can form chemical bonds through cross-linking, which can mask antigenic epitopes and lead to a weakening or disappearance of immunofluorescence staining signals.

[0040] Citrate buffer (typically pH 6.0) breaks cross-linked protein peptide chains by heating (e.g., in a microwave, pressure cooker, or water bath), re-exposing antigenic epitopes and restoring their ability to bind to antibodies.

[0041] It significantly improves staining signal intensity, reduces nonspecific background, and lowers the false negative rate.

[0042] 2. Maintain pH stability

[0043] Citrate buffer is a weakly acidic buffer system (pH 3-6). It can resist pH fluctuations during heating, ensuring the stability of the antigen retrieval environment and preventing the antigen epitope structure from being affected by pH changes.

[0044] After soaking, the sample was soaked in buffer 1 in the dark for 1 hour; the buffer 1 was prepared by dissolving 1 mL Triton X-100 in 100 mL 10×PBS buffer, and adding water to make up to 1 L, with pH=7.4.

[0045] Furthermore, a 10 mM citrate solution (specifically, a sodium citrate solution was used in this embodiment of the invention) was used, and the microwave treatment power was 50p. The microwave treatment was heated until the solution was close to boiling, i.e., 90-100°C.

[0046] Preferably, in step S4, the primary antibody is a ready-to-use goat serum primary antibody, incubated at 4°C in the dark for ≥48 hours.

[0047] The fluorescently labeled secondary antibody is a ready-to-use goat serum fluorescently labeled secondary antibody, incubated at 37°C in the dark for ≥70 min. During incubation, temperature and time are controlled to ensure sufficient binding of the antibody to the target protein.

[0048] After incubation with the primary antibody, wash with buffer 2; buffer 2 is prepared by dissolving 1 mL of Tween-20 in 100 mL of 10×PBS buffer, and then adding water to a final volume of 1 L, with a pH of 7.4.

[0049] Specifically, the following steps are included:

[0050] S301. Rinse the samples obtained in steps S2 and S3 slowly with 1 mL of ice-cold Carnoy fixative, air dry, then transfer to 10 mM citrate solution for soaking, microwave and soak for 3-5 min.

[0051] S302. Quickly remove the sample after the treatment in step S301 and soak it in buffer solution 1 in the dark for 1 hour.

[0052] S303. Take out the sample after step S302 and place it in a humidifying box. Add 40 μL of ready-to-use goat serum to the sample to block non-specific binding. Cover the sample and incubate at room temperature in the dark for 40 min.

[0053] S304. Remove the coverslip from the sample after step S303, add 40 μL of ready-to-use goat serum primary antibody to one side, cover with the coverslip and incubate at 4°C in the dark for 48 h.

[0054] S305. Remove the coverslip from the sample after step S304, wash three times with buffer 2 for 15 minutes each time, and preheat the humidification box at 37°C.

[0055] S306. Take out the sample after step S305 and place it in a preheated humidification 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.

[0056] S307. Remove the coverslip from the sample after step S306, wash three times with buffer 2 for 20 min each time, add 8 μL of DAPI (i.e., 4,6-diamidinyl-2-phenylindole, 5 μg / mL) to the center of the sample, and cover with a coverslip.

[0057] The beneficial effects of this invention are as follows:

[0058] (1) Through optimized enzymatic hydrolysis, the callosity protective layer on the outer layer of Lycoris pollen mother cells is effectively decomposed, thereby improving antibody permeability and enhancing the expression of fluorescence signals.

[0059] (2) Improved slide preparation and staining methods can better preserve the morphology and structure of cells and improve the stability and reproducibility of staining effects.

[0060] (3) It is applicable to a variety of Lycoris species, such as Lycoris radiata, Lycoris radiata and Lycoris radiata, and has a wide range of application prospects. Attached Figure Description

[0061] Figure 1 The results of immunofluorescence staining of HEI10, a localization homologous recombinant protein of Lycoris radiata meiotic cells in Example 1 of this invention, are observed under a fluorescence microscope. Scale bar = 10µm.

[0062] Figure 2The results of immunofluorescence staining of HEI10, a localization homologous recombinant protein of *Hypericum hudichotomum* meiotic cells in Example 2 of this invention, are observed under a fluorescence microscope. Scale bar = 10µm.

[0063] Figure 3 This image shows a comparison of immunofluorescence staining of SYN1 and HEI10 cells during meiosis in *Lycoris radiata* var. *chunxiaoensis* before the improvement of the immunofluorescence protein staining technique of this invention. Scale bar = 10µm.

[0064] Figure 4 The image shows a comparison of immunofluorescence staining of SYN1 and HEI10 cells during meiosis in *Lycoris radiata* cells after the improvement of the immunofluorescence protein staining technique of this invention. Scale bar = 10µm. Detailed Implementation

[0065] Example 1: Immunofluorescence staining technique for localization homologous recombinant protein HEI10 in Lycoris radiata meiotic cells

[0066] 1. Take flower buds of Lycoris radiata in the meiotic stage and immerse them in Carnoy's fixative (prepared by mixing anhydrous ethanol and acetic acid in a volume ratio of 3:1) for more than 24 hours. Remove the anthers and place them in Carnoy's fixative until they decolorize.

[0067] 2. Remove the anthers and wash them twice with ddH2O, then transfer them to a 10 mM sodium citrate solution and wash them once.

[0068] 3. Use a syringe to cut a portion of the anthers, place it on a glass slide, and squeeze to extract the pollen mother cells.

[0069] 4. Place the pollen mother cells and the broken anther tissue into a centrifuge tube, add 2.5% glucanase and soak for 1.5 hours for enzymatic hydrolysis.

[0070] 5. Add an appropriate amount of ddH2O to the centrifuge tube to wash the enzyme solution, centrifuge, and remove the supernatant.

[0071] 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 citrate buffer, pH 4.5) and soak for 5 hours for enzymatic hydrolysis.

[0072] 7. Add an appropriate amount of ddH2O again to wash the enzyme solution, centrifuge and remove the supernatant.

[0073] 8. Transfer the remaining precipitate onto a glass slide, add ddH2O and grind it into a paste, add 10-20 μL of 60% acetic acid solution, mix well and smear, dry and fix, and mark the position of the sample smear.

[0074] 9. Rinse the sample slowly with 1 mL of ice-cold Carnoy fixative, air dry, then transfer to 10 mM sodium citrate solution for immersion. Heat in a microwave oven (power p=50, solution close to boiling, temperature range 90-100℃) and immerse for 4 min.

[0075] 10. Quickly remove the sample and place it in Buffer 1 (1 mL Triton X-100 dissolved in 100 mL 10×PBS buffer, pH=7.4, then add deionized water to bring the volume to 1 L; the 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 bring the volume to 1 L and adjusting the pH to 7.4) and soak it in the dark for 1 hour.

[0076] 11. Place the sample in a humidified 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 min.

[0077] 12. Add 40 μL of ready-to-use goat serum primary antibody, cover with a slide, and incubate at 4°C in the dark for 48 min.

[0078] 13. Wash three times with buffer 2 (1 mL Tween-20 dissolved in 100 mL 10×PBS buffer, pH=7.4, and diluted to 1 L with deionized water), 15 min each time, and preheat the humidification box at 37°C.

[0079] 14. Place the sample in a preheated humidification box, add 40 μL of ready-to-use goat serum secondary antibody, and incubate at 37°C in the dark for 70 min.

[0080] 15. Wash three times with buffer 2 for 20 minutes each time. Add 8 μL of DAPI (5 μg / mL) to the center of the sample and cover with the slide.

[0081] 16. Apply nail polish evenly around the coverslip, let it air dry, and then store it in the dark at -20℃.

[0082] 17. Use a fluorescence microscope to observe and record the localization signal (HEI10 signal) of the target protein.

[0083] HEI10 protein is a key protein catalyzing the formation of type I homologous crossing over. In Arabidopsis, HEI10 signals are detected on chromosomes during the leptotene stage, at which time the HEI10 foci are numerous and small. Larger and 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 and brighter HEI10 foci, eventually forming approximately 10 HEI10 signals by diakinesis.

[0084] like Figure 1As shown, numerous HEI10 signals appeared on the pachytene chromosomes of *Lycoris radiata*, along with a few larger and brighter HEI10 signals. During diakinesis, the HEI10 signal significantly decreased, with only large and bright HEI10 signals observed on the chromosomes. This result is similar to phenomena observed in other species, demonstrating the reliability of our 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 crossing over occurred between each pair of homologous chromosomes in *Lycoris radiata*, ensuring at least one genetic exchange between homologous chromosomes and guaranteeing normal segregation of homologous chromosomes.

[0085] Example 2: Immunofluorescence staining technique for localized homologous recombinant protein HEI10 in *Hypericum humilis* meiotic cells

[0086] Take flower buds of *Lysimachia christinae* in the meiotic stage, and follow the same steps as in Example 1.

[0087] Fluorescence microscopy observation results as follows Figure 2 As shown, in *Lycoris radiata*, the HEI10 signal specifically appears on chromosomes, and its localization pattern is similar to that in *Lycoris radiata*. Numerous HEI10 focal points were observed during pachytene, and these focal points gradually aggregated and merged into larger HEI10 focal points.

[0088] Comparative Example 1: Immunofluorescence staining results of SYN1 and HEI10 signals in meiotic cells of *Lycoris radiata* before and after improvement of the technique.

[0089] 1. Take the flower buds of Lycoris radiata in the meiotic stage and immerse them in Carnoy's fixative (prepared by mixing anhydrous ethanol and acetic acid in a volume ratio of 3:1) for more than 24 hours. Remove the anthers and place them in Carnoy's fixative until they decolorize.

[0090] 2. Remove the anthers and wash them twice with ddH2O, then transfer them to a 10 mM sodium citrate solution and wash them once.

[0091] 3. Place in a centrifuge tube, 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 citrate buffer, pH 4.5) and soak for 3 hours for enzymatic hydrolysis.

[0092] 4. Add an appropriate amount of ddH2O to wash the enzyme solution, transfer the enzymatically hydrolyzed anthers to a glass slide, add ddH2O and grind into a paste, add 10-20 μL of 60% acetic acid solution, mix well and smear, dry and fix, and mark the position of the sample smear.

[0093] 5. Rinse the sample slowly with 1 mL of ice-cold Carnoy fixative, air dry, then transfer to 10 mM sodium citrate solution for immersion. Heat in a microwave oven (power p=50, solution close to boiling, temperature range 90-100℃) and immerse for 4 min.

[0094] 6. Quickly remove the sample and place it in Buffer 1 (1 mL Triton X-100 dissolved in 100 mL 10×PBS buffer, pH=7.4, then add deionized water to bring the volume to 1 L; the 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 bring the volume to 1 L and adjusting the pH to 7.4) and soak it in the dark for 40 min.

[0095] 7. Place the sample in a humidified 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 min.

[0096] 8. Add 40 μL of ready-to-use goat serum primary antibody, cover with a slide, and incubate at 4°C in the dark for 48 h.

[0097] 9. Wash three times with buffer 2 (1 mL Tween-20 dissolved in 100 mL 10×PBS buffer, pH=7.4, and diluted to 1 L with deionized water), 15 min each time, and preheat the humidification box at 37°C.

[0098] 10. Place the sample in a preheated humidifier box, add 40 μL of ready-to-use goat serum secondary antibody, and incubate at 37°C in the dark for 70 min.

[0099] 11. Wash three times with buffer 2 for 15 minutes each time. Add 8 μL of DAPI (5 μg / mL) to the center of the sample and cover with a cover plate.

[0100] 12. Apply nail polish evenly around the coverslip, let it air dry, and then store it in the dark at -20℃.

[0101] 13. Use a fluorescence microscope to observe and record the localization signals (SYN1 signal and HEI10 signal) of the target protein.

[0102] Fluorescence microscopy observation results as follows Figure 3 As shown, the SYN1 and HEI10 signals failed to form specific fluorescent focal points on the chromosomes of Lycoris radiata meiotic cells.

[0103] Example 3: Improved Immunofluorescence Staining Technique for SYN1 and HEI10 Signals in Meiotic Cells of Lycoris radiata

[0104] 1. Take the flower buds of Lycoris radiata in the meiotic stage and immerse them in Carnoy's fixative (prepared by mixing anhydrous ethanol and acetic acid in a volume ratio of 3:1) for more than 24 hours. Remove the anthers and place them in Carnoy's fixative until they decolorize.

[0105] 2. Remove the anthers and wash them twice with ddH2O, then transfer them to a 10 mM sodium citrate solution and wash them once.

[0106] 3. Use a syringe to cut a portion of the anther and place it on a glass slide to squeeze out the pollen mother cells (adding an extra step to the anther treatment).

[0107] 4. Place the pollen mother cells and the broken anther tissue into a centrifuge tube together, add 2.5% dextranase and soak for 1.5 hours for enzymatic hydrolysis (add a dextranase treatment step).

[0108] 5. Add an appropriate amount of ddH2O to the centrifuge tube to wash the enzyme solution, centrifuge, and remove the supernatant.

[0109] 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 citrate buffer, pH 4.5) and soak for 5 hours for enzymatic hydrolysis (extend the enzymatic hydrolysis time of mixed enzyme).

[0110] 7. Add an appropriate amount of ddH2O again to wash the enzyme solution, centrifuge and remove the supernatant.

[0111] 8. Transfer the remaining precipitate onto a glass slide, add ddH2O and grind it into a paste, add 10-20 μL of 60% acetic acid solution, mix well and smear, dry and fix, and mark the position of the sample smear.

[0112] 9. Rinse the sample slowly with 1 mL of ice-cold Carnoy fixative, air dry, then transfer to 10 mM sodium citrate solution for immersion. Heat in a microwave oven (power p=50, solution close to boiling, temperature range 90-100℃) and immerse for 4 min.

[0113] 10. Quickly remove the sample and place it in Buffer 1 (1 mL Triton X-100 dissolved in 100 mL 10×PBS buffer, pH=7.4, then add deionized water to bring the volume to 1 L; the 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 bring the volume to 1 L and adjusting the pH to 7.4). Soak in the dark for 1 hour (extend the buffer soaking time).

[0114] 11. Place the sample in a humidified 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 min.

[0115] 12. Add 40 μL of ready-to-use goat serum primary antibody, cover with a slide, and incubate at 4°C in the dark for 48 h.

[0116] 13. Wash three times with buffer 2 (1 mL Tween-20 dissolved in 100 mL 10×PBS buffer, pH=7.4, and diluted to 1 L with deionized water), 15 min each time, and preheat the humidification box at 37°C.

[0117] 14. Place the sample in a preheated humidification box, add 40 μL of ready-to-use goat serum secondary antibody, and incubate at 37°C in the dark for 70 min.

[0118] 15. Wash three times with buffer 2 for 20 minutes 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).

[0119] 16. Apply nail polish evenly around the coverslip, let it air dry, and then store it in the dark at -20℃.

[0120] 17. Use a fluorescence microscope to observe and record the localization signals (SYN1 signal and HEI10 signal) of the target protein.

[0121] Fluorescence microscopy observation results as follows Figure 4 As shown, the antibody binds well to the chromosome, and the SYN1 and HEI10 signals form specific fluorescent focal points on the chromosomes of Lycoris radiata meiotic cells.

Claims

1. A method for immunofluorescence staining of proteins from Lycoris radiata meiotic cells, characterized in that, Includes the following steps: S1. Obtaining Lycoris radiata meiotic cells: fix the flower buds of Lycoris radiata during meiosis, separate the anthers and squeeze out the pollen mother cells; S2, Two-stage enzymatic hydrolysis treatment: First stage: Soak pollen mother cells in dextranase solution for enzymatic hydrolysis; Second stage: After cleaning, soak in mixed enzyme solution for enzymatic hydrolysis; The enzymes in the mixed enzyme solution are cell helicase, cellulase and pectinase; The mass concentration of the dextranase solution is 2.5%; The mixed enzyme solution has a mass concentration of 0.3% and is dissolved in 10 mM citrate buffer at pH 4.5, wherein the ratio of cell helicase, cellulase and pectinase is 1:1:

1. S3. Fixation of cell smears after enzymatic digestion; S4. Incubate with primary antibody and fluorescently labeled secondary antibody sequentially; S5. After mounting, observe under a fluorescence microscope.

2. The method for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 1, characterized in that, The Lycoris species mentioned are selected from Lycoris radiata, Lycoris radiata var. chinensis, or Lycoris radiata var. hudixiao.

3. The method for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 1, characterized in that, In step S1, the fixative for fixing the flower buds of Lycoris radiata during meiosis is Carnos fixative; the Carnos fixative is prepared by mixing anhydrous ethanol and acetic acid in a volume ratio of 3:1; the fixation time is ≥24h; The meiotic period referred to is either the first meiotic division or the second meiotic division.

4. The method for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 1, characterized in that, In step S2, the soaking and enzymatic hydrolysis time with dextranase solution shall not be less than 1 hour, and the soaking and enzymatic hydrolysis time with mixed enzyme solution shall not be less than 4 hours.

5. The method for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 4, characterized in that, The soaking and enzymatic hydrolysis time with dextranase solution is 1.5 hours, and the soaking and enzymatic hydrolysis time with mixed enzyme solution is 5 hours.

6. The method for immunofluorescence staining of meiotic cell proteins based on Lycoris radiata according to claim 1, characterized in that, In step S3, when fixing the cell smear after enzymatic digestion, first add water to the digested cells and crush them, then add 10-20 μL of 60% acetic acid solution, mix well, dry and fix, and mark the position.

7. The method for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 1, characterized in that, Step S4, prior to incubation, also includes microwaving the fixed sample with citrate buffer, followed by soaking for 3-5 minutes; After soaking, the sample was soaked in buffer 1 in the dark for 1 hour; the buffer 1 was prepared by dissolving 1 mL Triton X-100 in 100 mL 10×PBS buffer, and adding water to make up to 1 L, with pH=7.

4.

8. The method for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 7, characterized in that, Use a 10 mM citrate solution, microwave at 50p, and heat to 90-100℃.

9. The method for immunofluorescence staining of meiotic cell proteins based on Lycoris radiata according to claim 1, characterized in that, In step S4, the primary antibody is a ready-to-use goat serum primary antibody, incubated at 4°C in the dark for ≥48 hours; 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 for immunofluorescence staining of Lycoris radiata meiotic cell proteins according to claim 9, characterized in that, After incubation with the primary antibody, wash with buffer 2; buffer 2 is prepared by dissolving 1 mL of Tween-20 in 100 mL of 10×PBS buffer, and then adding water to a final volume of 1 L, with a pH of 7.4.

Images