CN108507849B - Wheat root cell nucleus extraction method suitable for immunofluorescence analysis - Google Patents
Wheat root cell nucleus extraction method suitable for immunofluorescence analysis Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The invention discloses a wheat root cell nucleus extraction method suitable for immunofluorescence analysis, which comprises the following steps: (1) the method comprises the steps of (1) obtaining wheat radicles, (2) extracting cell nucleuses, (3) detecting the cell nucleuses by a DAPI fluorescent staining method, (4) performing immunofluorescence histochemistry analysis, and (5) performing statistical analysis. The extraction method provided by the invention is suitable for preparing the cell nucleus suspension liquid so as to facilitate subsequent relevant experiments of the sub-microscopic level cytology.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a wheat root cell nucleus extraction method suitable for immunofluorescence analysis.
Background
The immunofluorescence technique is based on the principle of antigen-antibody recognition reaction, firstly, the known antigen or antibody is marked with fluorescein to prepare fluorescent marker, then the fluorescent antibody (or antigen) is used as molecular probe to examine the corresponding antigen (or antibody) in cell or tissue. The detection of fluorescently labeled cells or tissues by fluorescence microscopy, and the determination of the nature and localization of the antigen or antibody, or the quantitative determination by means of related quantitative techniques [ SAINTE MARIE G.A Paraffin-Embedding Technique for students Employing immunological disorders [ J ]. Journal of biochemistry & cytology, 1962,10(3):250- & 256 ], are also known as immunohistochemistry.
At present, the experimental object applied to the plant immunohistochemistry is mainly a tissue specimen, and mainly comprises a paraffin section and a frozen section, wherein the paraffin section is the most common and basic method for manufacturing the tissue specimen, the tissue shape is well preserved, and the paraffin section can be archived for a long time, so that retrospective research is provided. However, if the nuclei are studied, in particular some modifications of nuclear histones in epigenetics, then tissue sections are not particularly suitable. First, the nucleus is only a fraction of the tissue cells but is critical relative to plant tissue. Nuclei in conventional tissue sections are individual relative to the entire sectionThe body is small and cannot be easily observed if immunofluorescence techniques are applied to some proteins in the nucleus. Secondly, the paraffin section sample preparation period is longer, the process is complicated, and because the paraffin section samples are fixed by formaldehyde, intracellular antigens form aldehyde bonds and carboxymethyl bonds to be sealed by partial antigenic determinants, and meanwhile, cross-linking occurs between proteins to conceal the antigenic determinants. Therefore, it is required that the staining be performed by first repairing or exposing the antigen, i.e., breaking the cross-links formed between the surrounding timing molecules, thereby restoring the original spatial morphology of the antigen [ Poplar frequency, improvement of conventional paraffin sectioning method [ J ]]Journal of biology, 2006,23(1): 45-46; research progress of immunofluorescence analysis of Huanghuawei, Dumet chrysanthemum [ J]Application of chemical engineering, 2007,36(4):394-]Histochemical Journal,1980,12(4): 381-403). Therefore, the current methods for applying immunofluorescence techniques to nuclear proteins mainly include extracting the nuclei of plant tissues to prepare a nuclear suspension, and further preparing the nuclear suspension into a nuclear patch, thereby completing the experiments of related immunohistochemistry [ Grumin a. use of immunofluorescent methods for students of plant nuclei [ J].Biulleten,1977；Beven A F,Simpson G G,Brown J W,et al.The organization of spliceosomal components in the nuclei of higher plants[J].Journal of Cell Science,1995,108:509；Bai X D,Correa V R,
T Y,et al.AY-WB phytoplasma secretes a protein that targets plant cell nuclei[J].Mol Plant Microbe Interact.2009,22(1):18-30】。
In order to prepare intact, non-aggregated and abundant nuclei for subsequent experimental analysis, researchers tried to explore the extraction of plant nuclei by different methods [ Ninghua. comparative study of different preparation methods of plant nuclei [ J ]. university of Master China (Kokai edition), 2009,43(2): 308-; pennella, Liu Fang, Song Guo Li, etc. a technology for efficiently extracting cotton cell nucleus [ J ] Anhui agricultural science, 2009,37(18):19-21 ]. Wheat, as the third grain crop in China, has been improved in genome information in recent years, and has made certain progress in gene editing technology [ which tiger, solemn, chenopodium, and the like. Therefore, the method has very important practical significance for the research of wheat nuclear protein, in particular nuclear histone closely related to epigenetics. However, due to the difference of structures and tissue sources of different plants, the preparation method suitable for other plant cell nucleuses is not suitable for wheat crops. The invention takes wheat radicle as material, and based on the research of predecessors [ Li H, Yan S, ZHao L, et al, Histone acetic assisted up-regulation of the cell wall related genes is absorbed in dissolved mail root rotating [ J ]. BMC Plant Biology,2014,14(1): 105; a wheat cell nucleus extraction method suitable for flow cytometry analysis is compared with a previous cell nucleus preparation method through improvement of an experiment method and experiment equipment, and according to the result of an immunofluorescence analysis experiment, the wheat cell nucleus extraction method suitable for the immunofluorescence analysis is compared with the previous cell nucleus preparation method, so that a complete cell nucleus suspension with good form and no aggregation is prepared, and a basic condition is provided for research on proteins in wheat cell nuclei by using the immunofluorescence analysis experiment.
Disclosure of Invention
The invention aims to provide a wheat root cell nucleus extraction method suitable for immunofluorescence analysis, which can reduce the time required by cell nucleus extraction, prolong the retention of protein activity in cell nuclei, increase the accuracy and success rate of subsequent target protein detection, reduce the concentration of impurities in cell nucleus suspension, reduce the influence of the impurities on the immunofluorescence experiment, increase the number of cell nuclei in the cell nucleus suspension, and facilitate the subsequent related statistical experiment and analysis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a wheat root cell nucleus extraction method suitable for immunofluorescence analysis comprises the following steps:
(1) obtaining young wheat roots:
sterilizing and washing semen Tritici Aestivi, placing in culture dish containing wet filter paper, germinating in dark (20-25 deg.C), transferring seedling to culture room for growth after 1 day of germination, supplementing nutrient solution for 2 times during growth period, collecting young root of semen Tritici Aestivi until 5 days,
the culture conditions of the seedlings in the culture room in the step (1) are as follows: at the temperature of 18-23 ℃, the relative humidity of 65-75 percent and the illumination for 12-14 h/d;
the nutrient solution is 1/2 Hoagland nutrient solution, and the formula is as follows: 2mM calcium nitrate (Ca (NO)3)2) 2.5mM potassium nitrate (KNO)3) 0.5mM ammonium Nitrate (NH)4NO3) 0.5mM potassium dihydrogen phosphate (KH)2PO4) 1mM magnesium sulfate (MgSO)4) 2.5 μ M Potassium iodide (KI), 50.2 μ M boric acid (H)3BO3) 50 μ M manganese sulfate (MnSO)4) 15 μ M Zinc sulfate (ZnSO)4) 0.52 μ M sodium molybdate (Na)2MoO4) 0.05 μ M copper sulfate (CuSO)4) 0.053. mu.M cobalt chloride (CoCl)2) 25 mu M of ethylene diamine tetraacetic acid monosodium ferric (EDTA FeNa) which is added by 20-30mL each time;
(2) extracting cell nucleuses:
taking 0.5g of wheat radicle obtained in the step (1), cutting the material into root segments of 4-6mm, placing the root segments in a culture dish on ice, adding 1mL of MgSO 24Buffer solution, cutting the material into small pieces with a new sharp double-sided blade, transferring the solution from the culture dish to a centrifuge tube through a simple self-made filter, centrifuging at 4 deg.C and 1500r/min for 8-12min, discarding supernatant, precipitating, and adding MgSO 24Resuspending the buffer solution to obtain a cell nucleus suspension;
the MgSO4The buffer had the following composition: 10mmol/L magnesium sulfate (MgSO)4) 50mmol/L potassium chloride (KCl), 5 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 3mmol/L dithiothreitol, 0.25% (v/v) TritonX-100, pH value 7.4-7.6, and the product can be prepared for use;
(3) detecting cell nucleus by DAPI fluorescent staining method:
dripping 20 mu L of the cell nucleus suspension prepared in the step (2) on a glass slide attached with polylysine to prepare a cell nucleus patch, airing at room temperature (the prepared cell nucleus patch can also be stored at 20 ℃ below zero for standby), dripping 20 mu L of DAPI dye solution on the glass slide to stain for 4-6min under the dark condition, removing the dye solution, washing for 5 times by using PBS (phosphate buffer solution) with the concentration of 0.01mM and the pH value of 7.2, covering a cover glass, and directly placing under a fluorescence microscope for observation and photographing;
(4) immunofluorescence histochemical analysis:
taking out the prepared cell nucleus patch, adding 50 μ L of 3% (m/v) BSA solution on a glass slide, covering a cover glass, and sealing at 37 ℃ for 1 h; removing the cover glass, washing with 0.01mM PBS (pH 7.2) solution for 3 times (4-6 min each time); adding 50 mu L of an antibody on the slide, covering the slide with a cover slip, and incubating for 12h at 4 ℃; removing the cover glass, washing with 0.01mM PBS (pH 7.2) solution for 3 times (4-6 min each time); adding 50 mu L of secondary antibody with FITC label on the slide in the dark, covering the cover slip, and incubating for 1h at 37 ℃; removing the cover glass, washing with 0.01mM PBS (pH 7.2) solution for 3 times (4-6 min each time); then, DAPI staining is carried out, signals are detected under a fluorescence microscope, and the results are recorded by photographing;
(5) statistical analysis:
selecting 5 pictures for related statistical experiments, and repeating the experiments for 3 times; data statistics of the diameter and the number of the cell nucleus utilizes software Image J, and 3 times of measurement is carried out to obtain an average value; relevant statistics two-tailed T-test analysis was performed using GraphPad Prism 7.00, with P <0.05 indicating significant differences.
A simple self-made filter consists of a 450-mesh nylon membrane and a sterilized 1.5ml centrifuge tube. The manufacturing method comprises the steps of cutting the nylon membrane into a circular sheet with the diameter of 2cm, folding the circular sheet into a leak shape, and directly putting the circular sheet into a centrifugal tube, wherein the three layers of filter membranes are close to the tube wall.
Through the technical scheme, the problem that the cell nucleus extracting solution is easy to leak when being filtered is mainly solved. In the process of extracting plant tissue cell nuclei, the required amount of extracting solution is very small and is only 1-2ml, but the existing cell filter screen is mainly used for preparing protoplasts and is not used for filtering the plant cell nuclei, so that compared with the extraction of the plant tissue cell nuclei, the cell filter screen is large in size and cannot be directly filtered to a 1.5ml centrifuge tube, and other glassware is needed for transferring, so that the collection of filtrate is inevitably influenced. The number of nuclei in the nuclear filtrate was significantly increased by using the modified nuclear filtration device (FIG. 2), from 60-75 (20. mu.L) to 330-350 (20. mu.L).
In addition, the aperture of the nylon mesh is changed from 500 meshes to 450 meshes, because the wheat crops are polyploid crops and have larger cell nucleuses than common higher plants, and the meristematic zone of the root of the plant has strong cell division capability and larger cell nucleuses compared with other parts in the same plant. Therefore, the aperture of the currently used filter screen cannot meet the requirement of extracting the wheat root cell nucleus, and as can be seen from fig. 2, the morphology of the wheat root cell nucleus extracted by the original method is incomplete, and cell fragments exist in the nuclear suspension.
Compared with the prior art, the invention has the advantages and beneficial effects that:
(1) the invention effectively solves the problem that the cell nucleus extracting solution is easy to leak when being filtered;
(2) the invention simplifies the method of the cell nucleus filtration, increases the efficiency of the cell nucleus filtration, obviously increases the number of the cell nucleus in the cell nucleus filtrate by using the improved cell nucleus filtration device (figure 2), and increases the number from 60-75 (20 mu L) to 330-350 (20 mu L);
(3) the method provided by the invention greatly reduces impurities in the cell nucleus suspension, maintains the activity of protein in the cell nucleus, and increases the success rate of the subsequent immunofluorescence experiment.
(4) The wheat root cell nucleus extracted by the method provided by the invention is complete in shape and clearly visible, and immunohistochemical analysis by taking nuclear histones H3 and H4 as target proteins shows that the fluorescence signal is obvious, the dyeing effect is good, and the positioning is accurate, so that the cell nucleus extraction method provided by the invention is suitable for performing immunofluorescence analysis on the wheat root cell nucleus.
Drawings
FIG. 1 is a schematic diagram of a homemade cell nucleus filter;
wherein A is a commonly used cell filter mesh screen; B. c is simple cell nucleus filter assembly. Scale 2cm (A, B, C).
FIG. 2 is a comparison of wheat root nucleus patches prepared by different nucleus extraction methods;
wherein A: observing the cell nucleus suspension by shearing method; b: observing the cell nucleus suspension by an enzymolysis method; c: cell nucleus DAPI staining by a cutting method; a1, B1, C1: cell nucleus DAPI staining by different extraction methods; scale 50 μm (a, B, C); scale 20 μm (a1, B1, C1); d: and (4) counting the number of the cell nuclei of the cell nucleus slide prepared by different cell nucleus extraction methods. Indicates that the group of statistics had significant differences from the other two groups (P < 0.001).
FIG. 3 shows immunofluorescence analysis of wheat radicle nuclear component proteins H3 and H4;
primary antibodies used in the experiments were Histone H3, Histone H4(Active Motif, Shanghai), FITC markers were excited using a 488nm laser, and pictures were fused using the software Image J. Scale 20 μm.
FIG. 4 shows the statistics of the mean gray values of fluorescence signals of H3 and H4 in wheat radicle endonucleosome
5 pictures are selected in a statistical experiment, the gray value measurement is carried out by using software Image J, and an average value is obtained after 3 times of measurement. Data are significantly different, 0.001< P <0.01
Detailed Description
Example 1
A wheat root cell nucleus extraction method suitable for immunofluorescence analysis comprises the following steps:
(1) obtaining young wheat roots:
the experimental material is wheat variety (Triticum aestivum L.) Huamai No. 8, the seeds are placed in a culture dish with moist filter paper for dark germination (20-25 ℃) after being disinfected and washed, seedlings are moved to a culture room for growth after 1 day of germination, nutrient solution is additionally poured for 2 times during the growth period, when the seedlings grow to the 5 th day, wheat radicles are taken for cell nucleus extraction experiment, and the culture conditions of the seedlings in the culture room are as follows: 18-23 ℃, 65-75% relative humidity and 12-14h/d illumination.
The nutrient solution is 1/2 Hoagland nutrient solution, and the formula is as follows: 2mM calcium nitrate (Ca (NO)3)2) 2.5mM potassium nitrate (KNO)3) 0.5mM ammonium Nitrate (NH)4NO3) 0.5mM potassium dihydrogen phosphate (KH)2PO4) 1mM magnesium sulfate (MgSO)4) 2.5 μ M Potassium iodide (KI), 50.2 μ M boric acid (H)3BO3) 50 μ M manganese sulfate (MnSO)4) 15 μ M Zinc sulfate (ZnSO)4) 0.52 μ M sodium molybdate (Na)2MoO4) 0.05 μ M copper sulfate (CuSO)4) 0.053. mu.M cobalt chloride (CoCl)2) 25 mu M disodium iron ethylenediaminetetraacetate (EDTA FeNa) is added by 20-30mL each time, and the preparation method is described in Deng X Y, Li J W, Yang C N, et al].Journal of Triticeae Crops,2009。
(2) Extracting cell nucleuses:
2.1 different cell nucleus extract formula
In recent years, plant cell nucleus extraction methods are mainly classified into two types according to the cell wall removal mode, namely a mechanical wall breaking method represented by a direct shearing method, namely MgSO4The buffer solution is a cell nucleus extracting solution used by a shearing method; an enzymolysis wall-breaking method using cell wall degrading enzyme as main action, the enzymolysis liquid is the extracting liquid for extracting cell nucleus. The formula is as follows:
(1)MgSO4buffer (see Zhang L, Qiu Z, Hu Y, et al.ABA treatment of stimulating mail sections VP1gene expression and selective promoter-associated tissue amplification [ J ] for formulation method]Physiologia Plantarum 2011,143(3):287-296) component: 10mmol/L magnesium sulfate (MgSO)4) 50mmol/L potassium chloride (KCl), 5 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 3mmol/L Dithiothreitol (DTT), 0.25% (v/v) TritonX-100, pH 7.4 or 7.5 or 7.6.
(2) The enzymatic hydrolysate (see Loureiro J, Rodriguez E,
J,et al.Comparison of four nuclear isolation buffers for plant DNA flow cytometry[J].Ann Bot,2006,98(3):679-689) the components are as follows: cellulase R-10(Yakult, Japan) (1.5%, w/v), pectinase Y-23(Yakult, Japan) (0.1%, w/v), 5mmol/L calcium chloride dihydrate (CaCl)2·2H2O), 5mmol/L mannitol, 0.5mmol/L potassium dihydrogen phosphate (KH)2PO4) 2mmol/L magnesium sulfate (MgSO)4) 3 mmol/L2-N morpholine ethane sulfonic acid (MES) with pH value of 5.5 or 5.6 or 5.7.
2.2 method for extracting multiple cell nuclei
2.2.1 the shear method (see Loureiro J, Rodriguez E,
J,et al.Comparison of four nuclear isolation buffers for plant DNA flow cytometry[J].Ann Bot,2006,98(3):679-689)
0.5g of wheat radicle obtained in the step (1) is added with MgSO prepared in the step 2.14Cutting the cell nucleus extract with sharp surgical scissors on ice, filtering with 500 mesh nylon net into centrifuge tube, standing and incubating at 4 deg.C for 10min, centrifuging at 1000r/min for 10min, discarding supernatant, precipitating, and adding MgSO 2.14The cell nucleus extracting solution is washed once and filtered by a nylon net with 500 meshes again, and the cell nucleus extracting solution is the cell nucleus suspension.
2.2.2 enzymolysis method
Taking 0.5g of wheat radicles obtained in the step (1), cutting the wheat radicles into small fragments by using a double-sided blade, placing the small fragments into the enzymolysis liquid prepared in the step 2.1, carrying out dark degradation for about 3 hours in an environment at 25 ℃, oscillating the small fragments once every 30 minutes (the ratio (m/v) of the wheat radicles to the enzymolysis liquid is 1: 5), then filtering the small fragments by using a 280-mesh nylon net, removing tissue fragments which are not completely hydrolyzed, transferring the filtrate into a centrifuge tube, centrifuging the filtrate at 800r/min for 2 minutes, collecting and suspending the filtrate by using PBS, adding 0.5% (v/v) Triton X-100 into the PBS suspension, incubating for 5-10 minutes, filtering the filtrate by using a 500-mesh nylon net, and obtaining a cell nucleus suspension.
2.2.3 cutting method
Combining the above two methods, and improving, collecting 0.5g of wheat radicle obtained in step (1), cutting into 4 or 5 or 6mm root segments, placing in ice culture dish, adding 1mL MgSO 2.14Buffer solution, cutting the material into small pieces with a new sharp double-sided blade, transferring the solution from the petri dish to a centrifuge tube through a simple self-made filter as much as possible, centrifuging at 4 deg.C 1500r/min for 8 or 10 or 12min, discarding the supernatant, precipitating, adding MgSO 2.1 prepared in step 2.14And (4) resuspending the cell nucleus extracting solution to obtain cell nucleus suspension.
(3) Detecting cell nucleus by DAPI fluorescent staining method:
and (3) dripping 20 mu L of the cell nucleus suspension prepared in the step 2.2.3 onto a glass slide attached with polylysine to prepare a cell nucleus patch, drying the cell nucleus patch at room temperature (the prepared cell nucleus patch can also be stored at 20 ℃ below zero for standby), dripping 20 mu L of DAPI dye liquor (Biyunshi, C1006) on the glass slide to stain for 4 min or 5 min or 6min under the condition of keeping out of light, removing the dye liquor, washing the cell nucleus patch 5 times by PBS (0.01mM, pH 7.2), covering a cover glass, and directly placing the cell nucleus patch under a fluorescence microscope (Olympus BX-53) for observation and photographing.
(4) Immunofluorescence histochemical analysis:
taking out the prepared cell nucleus patch, dripping 50 mu L of 3% (m/v) BSA on a glass slide, covering a cover glass, and sealing at 37 ℃ for 1 h; cover slips were removed and washed 3 times with PBS solution (0.01mM, pH 7.2) for 4 or 5 or 6min each; adding 50 mu L of an antibody on the slide, covering the slide with a cover slip, and incubating for 12h at 4 ℃; removing the cover glass, and washing with PBS solution for 3 times, each time for 4 or 5 or 6 min; adding 50 mu L of secondary antibody with FITC label on the slide in the dark, covering the cover slip, and incubating for 1h at 37 ℃; removing the cover glass, and washing with PBS solution for 3 times, each time for 4 or 5 or 6 min; DAPI staining was then performed, signals were detected under a fluorescent microscope (Olympus BX-53), and the results were recorded by photography.
(5) Statistical analysis:
selecting 5 pictures for related statistical experiments, and repeating the experiments for 3 times; data statistics of the diameter and the number of the cell nucleus utilizes software Image J, and 3 times of measurement is carried out to obtain an average value; relevant data was analyzed by two-tailed T-test using GraphPad Prism 7.00, with P <0.05 indicating significant differences.
Example 2
Optimization and improvement of cell nucleus filtering method
The method for filtering the cell nucleus is a key test in one step in the preparation process of the cell nucleus suspension, and the test aims at the improvement and optimization of the extraction method of the cell nucleus at the root part of the wheat and mainly aims at the improvement of the traditional complicated and inconvenient cell nucleus filtering method. Firstly, a filtering device is replaced by a simple filter (figure 1C) which can be made by oneself from a cell mesh screen (figure 1A) used for filtering cell nucleuses, the required materials are a nylon membrane with 450 meshes and a sterilized 1.5ml centrifuge tube (figure 1B), the nylon membrane is cut into a circular sheet with the diameter of 2cm, the circular sheet is folded into a leak shape and is put into the centrifuge tube, and the positions of three layers of filter membranes are close to the tube wall (figure 1C).
Compared with the extraction of plant tissue cell nucleuses, the existing cell filter screen is large in size, cannot be directly filtered to a centrifugal tube of 1.5ml, and is inconvenient to operate. By using the improved nuclear filtration device, the nuclear filtration step is simplified, and the number of the nuclei in the nuclear filtrate is obviously increased from 60-75 (20 muL) to 330-350 (20 muL). In addition, the aperture of the nylon mesh is changed from 500 meshes to 450 meshes because the cell nucleus of the root of the wheat crop is larger than that of other parts of the common plant. Extraction of wheat root nuclei by the original method resulted in morphological incompleteness, leaving nuclear fragments in the nuclear suspension (FIG. 2A, A1).
In general, the improved cell nucleus filtering method simplifies the filtration of cell nuclei, is simpler and more convenient to operate, increases the cell nucleus filtering efficiency, reduces impurities in cell nucleus suspension, and maintains the integrity of the cell nuclei.
Example 3
Comparing the effect of extracting the wheat radicle nucleus by three different extraction methods
The results of extracting wheat radicle cell nucleus by different methods are shown in figure 2. As can be seen from the figure, the amount of impurities in the cell nucleus slide prepared by the shearing method is large, and cell fragments are not easily distinguished from cell nuclei (FIG. 2A). In addition, some nuclei have incomplete morphology and multiple nuclei are present in clusters that stick together and are not easily separated (FIG. 2A 1). However, the number of nuclei in the cell nucleus suspension obtained by the shearing method was the largest (FIG. 2D).
The process of preparing the cell nucleus suspension by the enzymolysis method has a long period, and the activity of the cell nucleus is not suitable to be ensured. And the number of nuclei extracted by this method was minimal (fig. 2D). However, the nuclei prepared by this method were intact in morphology and had fewer impurities (FIG. 2B 1).
The wheat radicle nucleus patch prepared by the cutting method has more nuclei, less impurities and only a very small amount of cell fragments in the visual field (figure 2C). The morphology of the cell nucleus is observed to be in a full circle or ellipse under a microscope after DAPI staining (figure 2C1), the cell nucleus extraction process is simple and convenient, and the preparation of the cell nucleus suspension is convenient and quick. Therefore, immunofluorescence histochemistry experiments of wheat radicle nuclear inner histones H3, H4 were performed using the cell nuclear slide prepared by this method.
Results of immunofluorescence analysis of wheat radicle endonucleo-histones H3 and H4 are shown in the figure (FIG. 3). The nuclear histones H3 and H4 immunohistochemical experiments are carried out on the wheat root cell nuclear slide prepared by the cutting method, and the cell nuclear slide after the immunohistochemical experiments are placed under a fluorescence microscope (100 times oil scope) for observation under the dark room condition. The shape of the cell nucleus is complete and clearly visible, the fluorescence signal of the target protein FITC is obvious, the overlapping of the fluorescence signal can be clearly observed after fusion, and simultaneously, the diameter and the number of the cell nucleus and the fluorescence signal can be quantitatively processed by using software such as Image J and the like. In this experiment, the average diameter of the wheat rootlet nuclei in the nucleus suspension prepared by the cleavage method was 26 μm, the average number was 340 (20. mu.l), and the quantitative analysis of the fluorescence signal of the target protein was shown in FIG. 4.
In previous nuclear suspension preparations, the efficiency of nuclear filtration was one of the key factors in the number and integrity of nuclei in nuclear suspensions [ gualbetti G,
J,Macas J,et al.Preparation of pea(Pisum sativum,L.)chromosome and nucleus suspensions from single root tips[J].Theoretical&applied Genetics,1996,92(6):744-751 ]. The size and material of the pore diameter of the filter screen for filtering cell nucleus have been studied in more detail by scholars at home and abroad [ Gualberti G,
J,Macas J,et al.Preparation of pea(Pisum sativum,L.)chromosome and nucleus suspensions from single root tips[J].Theoretical&applied Genetics,1996,92(6): 744-751; preparation of Taxus chinensis nuclear suspension in flow cytometry assay [ J]Journal of university of forestry in southwest (science of Nature), 2013,33(1): 97-100), the current general nuclear filter screen has a pore size of 500 meshes, and the material is also improved from the original stainless steel to the currently used nylon screen [ Ninghua, comparative study of different preparation methods of plant nuclei [ J]The university of Master and university in China (own edition), 2009,43(2): 308-. However, the use of the conventional cell filtration screen (fig. 1A) for filtering the extract into the centrifuge tube is inconvenient, and the extract may leak out to reduce the amount of extracted cell nuclei. In addition, for wheat, which is a higher crop, the size of the nuclei is larger than that of other plants, and the size of the nuclei in the roots is larger than that of other parts of the plant (such as leaves) [ Ali Arak University Rezaei, major Arak University DNA fragmentation and change of nuclei in saline cells of wheat root rot [ J].International Journal of Forest Soil&Erosion, 2013; liujian, Fangfang, Xubaohua, etc. Electron microscopy of the structure of the inclusion body in the nucleus of the meristematic nucleus of the root end of common wheat [ J]Analytical instrument 2011(2):54-56 ], therefore, the aperture of 500 meshes is not necessarily suitable for the extraction of wheat root cell nuclei. The invention aims at the improvement of the extraction method of wheat root cell nucleuses, and firstly, a simple filter which can be made by self is designed for filtering cell nucleus extracting solution (figure 1C). So, when using the pipettor with the cell nucleus extract, when following the centrifuge tube pipe wall and slowly pouring into the filter, can avoid the omission of extract, increase the quantity of cell nucleus in the suspension.
For extraction of nuclei suitable for immunohistochemical experiments, this requires the preparation of a suspension of nuclei in which a large number of intact nuclei are present and which do not aggregate. Therefore, various components are added to the extraction buffer in order to ensure the morphological and functional integrity of the nuclei. Inorganic salt (KCl) energyCapable of maintaining the ionic strength, Mg, in the buffer2+Can stabilize chromatin [ Lee H, Lin T.isolation of plant core capable for flow cytometry from nuclear catalysis tissue by use of a transduction column [ J ]]Plant Molecular Biology Reporter,2005,23(1):53-58, DTT is able to eliminate the effect of phenolic mixtures on the staining of nuclear DNA, protect chromatin proteins [ Jinliang, Zhanxianyin, Xuehong ] comparison of the resolution effect of isolation buffers on nuclear suspension DNA in rice [ J]Zhejiang agricultural journal, 2007,19(2):93-96 ], Triton X-100 is a non-ionic detergent capable of facilitating the release of the nucleus from the cell into solution while maintaining the relative stability of the pH in the buffer using Tris base. During the enzymatic extraction of the nuclei, the nuclei are finally resuspended in a PBS solution, the pH of which differs considerably from the enzymatic solution used for the extraction, and changes in the pH are liable to lead to fragmentation of the nuclei in the young roots of wheat [ Masuda A, Oyamada M, Nagaoka T, et al]Brain Research,1998,807(1-2):70 ], which should be the direct cause of the small number of wheat radicle nuclei in the nuclear suspension. Thus, the improvement of extracting wheat root cell nucleus retains MgSO in shearing method4Use of a buffer. In addition, the tissue is not easily cut too much when the material is processed, and impurities (Sun Yongxing, plum blossom, Weixiaoli, etc.) except the cell nucleus are prevented from being mixed in the filtrate during filtering]Journal of biology, 2012,29(3): 88-91; study on influence of UV-B radiation and He-Ne laser irradiation on wheat cell apoptosis]University of shanxi professor, 2012). The material is treated with surgical scissors in the shearing process to cause excessive cell debris, so that the material is replaced with a new sharp blade in the modified cutting process, and the material is cut into small pieces with the sharp blade to allow the nuclei of the root cells to flow out of the extraction solution, so that the finally prepared nucleus suspension does not have excessive cell debris impurities (fig. 2C).
In the invention, wheat root cell nuclei are extracted by an improved cutting method, and are subjected to immunohistochemical staining, then the wheat root cell nuclei are detected under a fluorescence microscope, and then data processing and quantitative analysis are performed on images by using related software, wherein the results are shown in fig. 2 and fig. 3. The wheat root cell nucleus extracted by the improved cutting method in the experiment is complete in shape and clearly visible, and immunohistochemical analysis by taking nuclear histones H3 and H4 as target proteins shows that the fluorescence signal is obvious, the dyeing effect is good, the positioning is accurate, and the cell nucleus extraction method is suitable for performing immunofluorescence analysis on the wheat root cell nucleus.
Claims (1)
1. A wheat root cell nucleus extraction method suitable for immunofluorescence analysis comprises the following steps:
(1) obtaining young wheat roots:
sterilizing and washing semen Tritici Aestivi, placing in culture dish containing wet filter paper at 20-25 deg.C, germinating for 1 day, transferring seedling to culture room for growth, supplementing nutrient solution for 2 times during growth period, collecting young root of semen Tritici Aestivi when young seedling grows to 5 days,
the culture conditions of the seedlings in the culture room are as follows: at the temperature of 18-23 ℃, the relative humidity of 65-75 percent and the illumination for 12-14 h/d;
the nutrient solution is 1/2 Hoagland nutrient solution, and the formula is as follows: 2mM calcium nitrate, 2.5mM potassium nitrate, 0.5mM ammonium nitrate, 0.5mM monopotassium phosphate, 1mM magnesium sulfate, 2.5. mu.M potassium iodide, 50.2. mu.M boric acid, 50. mu.M manganese sulfate, 15. mu.M zinc sulfate, 0.52. mu.M sodium molybdate, 0.05. mu.M copper sulfate, 0.053. mu.M cobalt chloride and 25. mu.M monosodium ferric ethylenediamine tetraacetate, wherein 20-30mL of each addition is carried out;
(2) extracting cell nucleuses:
taking 0.5g of wheat radicle obtained in the step (1), cutting the material into root segments of 4-6mm, placing in an ice culture dish, adding 1mM MgSO4Buffer solution, cutting the material into small pieces with a double-sided blade, allowing the cell nucleus of root cell to flow out of the extractive solution, transferring the solution from the culture dish to a centrifuge tube via a filter, centrifuging at 4 deg.C for 8-12min at 1500r/min, discarding the supernatant, adding MgSO (MgSO) into the precipitate4Resuspending the buffer solution to obtain a cell nucleus suspension;
step (ii) of(2) MgSO as described in (1)4The buffer had the following composition: 10mmol/L magnesium sulfate, 50mmol/L potassium chloride, 5 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 3mmol/L dithiothreitol, 0.25 percent TritonX-100 in volume ratio, and 7.4-7.6 of pH value, and the mixture is ready for use;
the filter in the step (2) consists of a nylon membrane with 450 meshes and a sterilized 1.5mL centrifuge tube, and the manufacturing method comprises the steps of cutting the nylon membrane into a wafer with the diameter of 2cm, folding the wafer into a leak hole shape, and directly placing the wafer into the centrifuge tube, wherein the three layers of filter membranes are close to the tube wall;
(3) detecting cell nucleus by DAPI fluorescent staining method:
taking 20 mu L of the cell nucleus suspension prepared in the step (2), dripping the cell nucleus suspension on a glass slide attached with polylysine to prepare a cell nucleus patch, drying the cell nucleus patch at room temperature, dripping 20 mu L of DAPI dye solution on the glass slide to stain for 4-6min under the condition of keeping out of the sun, removing the dye solution, washing the cell nucleus patch for 5 times by using PBS (phosphate buffer solution) with the concentration of 0.01mM and the pH value of 7.2, covering a cover glass, directly placing the cell nucleus patch under a fluorescence microscope to observe and take a picture;
(4) immunofluorescence histochemical analysis:
taking out the prepared cell nucleus patch, adding 50 mu L of BSA solution with the mass volume ratio of 3% on a glass slide, covering the glass slide, and sealing for 1h at 37 ℃; removing the cover glass, washing with 0.01mM PBS (pH7.2) solution for 3 times (4-6 min each time); adding 50 mu L of an antibody on the slide, covering the slide with a cover slip, and incubating for 12h at 4 ℃; removing the cover glass, washing with 0.01mM PBS (pH7.2) solution for 3 times (4-6 min each time); adding 50 mu L of secondary antibody with FITC label on the slide in the dark, covering the cover slip, and incubating for 1h at 37 ℃; removing the cover glass, washing with 0.01mM PBS (pH 7.2) solution for 3 times (4-6 min each time); then, DAPI staining is carried out, signals are detected under a fluorescence microscope, and the results are recorded by photographing;
(5) statistical analysis:
selecting 5 pictures, and repeating the experiment for 3 times; data statistics of the diameter and the number of the cell nucleus utilizes software Image J, and 3 times of measurement is carried out to obtain an average value; relevant statistics two-tailed T-test analysis was performed using GraphPad Prism 7.00, with P <0.05 indicating significant differences.
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| CN101782582B (en) * | 2010-01-11 | 2012-11-07 | 天津师范大学 | Immunofluorescence method for detecting nucleolus fibrin positioning under heavy metal stress |
| CN102012425A (en) * | 2010-12-29 | 2011-04-13 | 南开大学 | New immuno-fluorenscence labelling method |
| CN102288471B (en) * | 2011-07-27 | 2013-06-12 | 上海交通大学医学院附属仁济医院 | Immunofluorescence staining method for suspension cells |
| WO2014177700A1 (en) * | 2013-05-02 | 2014-11-06 | Universite D'aix-Marseille | Indirect immunofluorescence method for detecting antinuclear autoantibodies. |
| CN103869082B (en) * | 2014-03-18 | 2016-02-03 | 浙江大学 | The method of immuno-fluorescence assay HLA-DR expression assessment PBMC antigen presentation capability |
| CN105784573A (en) * | 2016-03-07 | 2016-07-20 | 天津师范大学 | Method for analyzing wheat root tip cell cycle through flow cytometry |
| CN106544411A (en) * | 2016-08-27 | 2017-03-29 | 华中农业大学 | A kind of method for identifying mammal y sperm typing using mark analysis of protein |
| CN107132344A (en) * | 2017-03-16 | 2017-09-05 | 国家烟草质量监督检验中心 | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis aldehyde material |
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