CN101782573B - Immunofluorescence method for detecting nucleolus C23 protein positioning under heavy metal stress - Google Patents
Immunofluorescence method for detecting nucleolus C23 protein positioning under heavy metal stress Download PDFInfo
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Abstract
The invention discloses an immunofluorescence method for detecting plant cell nucleolus C23 protein positioning under heavy metal stress. The method comprises the following steps: cutting plant root tip meristem cells after heavy metal treatment; fixing the cells in 4 percent paraformaldehyde for 1 hour; adopting enzyme fluid to perform enzymolysis; sucking about 0.1 millimeter of sample solution with a dropper; uniformly smearing the sample solution on a glass slide to disperse the sample solution into single cells; naturally airing the obtained product after labeling; soaking the obtained product in 1 percent TritonX-100 for 15 minutes; dropping 15 mu l of a first antibody prepared; covering the obtained product with a cover slide; incubating the obtained product for 1 hour in a warm box at 37 DEG C; dropping 15 mu l of a second antibody prepared; dropping 15 mu l of DAPI after treatment; dropping 5 mu l of anti-quenching agent on a glass slide material; covering the obtained product with the cover slide; sealing the periphery of the cover slide with nail polish; and observing the obtained product under a fluorescence microscope after 1 hour. The detection method has the characteristics of high specificity, accurate experimental result, great increase in the number of observation cells, and the like. The method provides a precise detection method for studying the cell poisoning mechanism of heavy metal, and the detection method has broad application prospects.
Description
Technical field
The invention belongs to cytobiology technology field, relate to the localization method that under a kind of fast detecting heavy metal stress, nucleolus protein changes.Say more specifically a kind of immunofluorescence method that detects vegetable cell kernel C23 protein positioning under heavy metal stress.The method provides new technical support for studying heavy metallic poison vegetable cell mechanism.
Technical background
Along with the development of the modern industry, the problem of environmental pollution causing thus becomes increasingly conspicuous.In addition the unreasonable of chemical fertilizer and agricultural chemicals used, the discharge of " three wastes " and domestic waste, accelerate the pollutant entered environment by all means of heavy metal (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Zn etc.), and endanger animal and human's body health by food chain, cause the further deterioration of atmosphere and quality of water environment.At present, global Heavy Metal Pollution is day by day serious, how to control and to alleviate pollution and the harm of heavy metal to environment, has become the hot subject that environment, soil and related discipline scientists are paid close attention to.Some researchs have shown that low-concentration heavy metal has positive spread effect to the growth of plant, but too high when heavy metal concentration in environment, the growth of plant are produced to toxic action, cause the variation of plant physiology, biochemistry; Suppress the absorption of Plant To Nutrient element; Damaging cells structure; Affect plant normal growth metabolism.
Kernel (nucleolus) is eukaryotic interphase nucleus inner height structure closely, and it is that rDNA transcribes the place of assembling with ribosomal subunit.Kernel is not only the center of the interior communication of cell and rRNA processing, along with cell cycle on cell proliferation and an old and feeble important regulating and controlling effect; In general, the chemical composition of kernel is mainly made up of DNA, RNA, protein and enzyme etc.Wherein account for 80% of dry weight with protein.RNA accounts for 10% of dry weight, and many and protein bound, exists with the form of nucleoprotein.Utilize proteomics method to identify nucleolin in 350, the function of these protein and kernel is closely related.From Physiology and biochemistry, molecule and cell biology means, research heavy metal is synthetic on nucleolus protein, the impact of distribution and function, has caused association area scientist's concern.
The inventor utilize this technical research different heavy metals (Al, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Zn) coerce the impact on plant seedlings root-tip cells kernels such as broad bean, garlic, onion and corns, these results of study show: heavy metal stress can damage nucleolar structure, cause in kernel silver to dye protein substance excessive in tenuigenin.Other scholars, as Li Xiaoling, Zhang Yixian, process mung bean and barley with variable concentrations nickel chloride, and the method for dying by silver is also observed Ni2+ and can be induced mung bean and Barley Roots tip Cells nucleolar silver staining protein body quantity obviously to increase.Therefore, utilize the impact of silver-colored dyeing technique research counterweight metal pair vegetable cell kernel structure, there is certain scientific value.But, silver dyeing technique can only be identified after heavy metal stress, be nucleolin from the excessive Ag-NOR to tenuigenin of nucleus, can not identify it is the impact which kind nucleolin is subject to heavy metal stress, further investigation heavy metallic poison mechanism is had to certain limitation.
In the last few years, the development of fluoroimmunoassay technology was particularly rapid, in life science, had been widely used for measuring the aspect researchs such as growth factor, protein positioning, foranalysis of nucleic acids, neurotransmitter.Nucleolin C23 is one of important protein component of eukaryotic kernel, and in the ribosomal biosynthesizing of regulation and control, with ripe, cell proliferation, growth, embryo's generation, cytokinesis, chromatin copy with the process such as the generation of kernel and plays a significant role.But, utilize immunofluorescence technique research heavy metal stress the function of C23 protein in kernel periodic process and the impact of dynamic change to be there is not yet to the report of pertinent literature.
Summary of the invention
The object of this invention is to provide a kind of immunofluorescence method that detects vegetable cell kernel C23 protein positioning under heavy metal stress, under research environment stress in cell protein positioning and variation characteristic simple and easy, method is accurately and rapidly provided.
The inventor, in conjunction with laboratory previous work, has invented a kind of immunofluorescence method that detects vegetable cell kernel C23 protein positioning under heavy metal stress.The cardinal principle of this technology is that immunological method (antigen-antibody specific bond) and fluorescence labeling technology are combined, the method that research differential protein antigen distributes in cell.The fluorescence of sending out due to fluorescein can detect under fluorescent microscope, thereby determines character, the location of antigen or antibody, and utilizes quantitative technique to measure content.The reform of the tabletting method particularly the present invention relates to, can improve observation of cell number, applicable to other RESEARCH ON CELL-BIOLOGY.For achieving the above object, the invention provides following technical scheme:
An immunofluorescence method that detects vegetable cell kernel C23 protein positioning under heavy metal stress, is characterized in that, is undertaken by following step:
(1) cut heavy metal plant root tip meristematic cell after treatment, in 4% paraformaldehyde, fix 1-2h; Wash through PBS damping fluid;
(2) adopt 2.5% cellulase+2.5% pectase enzyme liquid in 37 DEG C of incubator enzymolysis; Compressing tablet after the washing of PBS damping fluid;
(3) plant root tip is gone in centrifuge tube, drip PBS damping fluid, hand concussion, to most cell free states, is drawn about 0.1-0.2ml sample liquid with dropper, is evenly applied on microslide and is dispersed into individual cells, for subsequent use after natural air drying after mark;
(4) microslide (3) being obtained is placed on 1%TritonX-100 and soaks 15-20 minute; Wash through PBS damping fluid;
(5) on the slide glass obtaining in (4), splash into the primary antibodie that 15-20 μ l prepares, covered, 37 DEG C of incubation 1h-1.5h; Wash through PBS damping fluid again;
(6) what on the slide glass again (5) being obtained, splash into that 15-20 μ l prepares is two anti-, covered, 37 DEG C of incubation 45min-1h; Wash through PBS damping fluid;
(7) 15-20 μ l DAPI, covered will be dripped again on the microslide of (6); Wash through PBS damping fluid;
(8) blot unnecessary PBS with filter paper, drip 5-10 μ l anti-quencher on microslide material, covered, seals cover glass surrounding with nail polish, after 1-2h at fluorescence microscopy Microscopic observation.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, wherein the enzymolysis time used refers in step (2): divide equally and scatter if observe most cells in the time of microscopy, become the spherical bioplast of one deck, wherein the tenuigenin of a small amount of cell disintegrates, only when remaining nucleus, finish enzymolysis.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, wherein the compressing tablet described in step (2) refers to: after enzymolysis, directly sample is made to suspending liquid state, then, drop in equably on microslide, do not cover cover glass, for subsequent use after natural air drying.
PBS damping fluid of the present invention is washed and is referred to and adopt PBS damping fluid to wash 3 times, each 10-15 minute.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, primary antibodie is wherein: mouse-anti C23 monoclonal antibody, its concentration is 0.2mg/ml.Two resist and are: TRITC-mountain its concentration of sheep anti-mouse igg is 1.5mg/ml.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, wherein the concentration of DAPI (4,6-diamines-2 Phenylindole-bis-hydrochloric acid) is 1 μ g/ml.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, 1%Triton X-100 is wherein Triton X-100 (Qu Latong).
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, anti-quencher is wherein 10 × 1ml.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, heavy metal is wherein Al, As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb or Zn.Preferably As, Cd, Co, Al, Cr, Cu, Hg, particularly preferably Al, Cd and Pb.
The immunofluorescence method of vegetable cell kernel C23 protein positioning under detection heavy metal stress of the present invention, wherein said vegetable cell comprises: the variation of nucleolus C 23 protein in broad bean, onion, corn and Onion root-tip cell.
The time that enzymolysis of the present invention is required, the per sample concrete condition of enzymolysis and determining.According to the inventor's experience, in the time of microscopy, observe most cells and divide equally and scatter, become the spherical bioplast of one deck, wherein the tenuigenin of a small amount of cell disintegrates, and only finishes enzymolysis, the best results of acquisition when remaining nucleus.After enzymolysis, directly sample is made to suspending liquid state, then, dropped in equably on microslide, need not cover cover glass, for subsequent use after natural air drying.This method has deducted cover plate and two steps of peel in conventional compressing tablet, has avoided the damage to cell in the operation of these two steps.
The more detailed experimental procedure of the present invention is as follows:
Taking plant root tip as experiment material, in the time of the about 1.5cm of root growth length left and right, carry out different heavy metal stress experiments.
(1) cut heavy metal apical meristem cell after treatment, in 4% paraformaldehyde, fix 1-2h.
(2) in PBS damping fluid (pH7.0), wash (each 10 minutes) 3 times.
(3) 2.5% cellulase+2.5% pectases, 37 DEG C of incubator enzymolysis, determine the time according to cell enzymolysis situation.
(4) PBS damping fluid is washed (each 10 minutes) 3 times.
(5) tip of a root is gone in the centrifuge tube of 0.5ml, drip PBS damping fluid a small amount of (suitable sample how many and determine), lid centrifuge tube lid, hand concussion is most cell free states extremely.Draw about 0.1-0.2ml sample liquid with dropper, be evenly applied on slide glass and be dispersed into individual cells, do not cover cover glass, the n.s. face mark with marking pen at slide glass, for subsequent use after natural air drying.
(6) section that will mark is placed on 1%TritonX-100 Triton X-100 (Qu Latong) and soaks 20 minutes.
(7) PBS damping fluid is washed (each 10 minutes) 3 times.
(8) blot unnecessary PBS damping fluid with filter paper, drip that (primary antibodie (being combined with C23 nucleolin) that 20 μ l) prepare is to the sample place of microslide, covered, 37 DEG C of incubation 1h-1.5h.It is emphasized that: the packing as early as possible of the antibody of purchase, packing can farthest reduce the infringement of multigelation antagonist activity, has also reduced owing to repeatedly drawing the possibility of pollution that antibody causes from same pipe simultaneously.
(9) in PBS damping fluid, wash (each 10 minutes) 3 times.
(10) blot unnecessary PBS damping fluid with filter paper, drip (two anti-(with the fluorescein of primary antibodie structure) that 20 μ l) prepare to the sample of microslide, covered.37 DEG C of incubation 45min-1h.
(11) in PBS damping fluid, wash (each 10 minutes) 3 times.
(12) DAPI (DNA dyestuff) dyeing: blot unnecessary PBS damping fluid with filter paper, drip that (20 μ l) DAPI cover microslide.
(13) in PBS damping fluid, wash (each 10 minutes) 3 times.
(14) blot unnecessary PBS with filter paper, drip that (l) anti-quencher is on microslide material for 10 μ, and covered, seals cover glass surrounding with nail polish, can be at fluorescence microscopy Microscopic observation after 1h.
(15) fluorescence microscope: blue light 450～490nm for nucleolus C 23 albumen (TRITC) excites and is fluorescent red-orange.DNA (nucleus and chromosome) excites and is blue-fluorescence with ultraviolet light 355～425nm (DAPI).
Configuration and the preservation of main agents that the present invention uses:
(1) primary antibodie: mouse-anti C23 monoclonal antibody (mouse monoclonal anti-C23 antibody), purchased from SantaCruz (U.S.) company.Primary antibodie concentration is 0.2mg/ml, each 1ml that is packaged as.After purchase, be sub-packed in 1.5ml centrifuge tube-20 DEG C of preservations by every pipe 10 μ L.Treat that the used time thaws.Before using, be diluted to 1.5ml with PBS damping fluid (pH7.4) and get final product (being equivalent to dilute 150 times), 4 DEG C of preservations.
(2) two is anti-: TRITC-mountain sheep anti-mouse igg (Tritc-conjugated rabbit anti-Mouse IgG (H+L)), and purchased from Jackson (U.S.) company.Imported with original packaging is freeze-dried powder, 2.0ml; Import is packed as liquid, 0.1ml (separately adding 0.1ml glycerine).Be sub-packed in 0.5ml centrifuge tube by every pipe 10 μ L ,-20 DEG C keep in Dark Place.Treat that the used time thaws, being diluted to 0.5ml with PBS buffer (pH7.6) can (being equivalent to dilute 50 times), and 4 DEG C keep in Dark Place stand-by
(3) DAPI (4,6-diamines-2 Phenylindole-bis-hydrochloric acid) DAPI mainly dyes DNA in nucleic acid, and storage liquid is made into 1mg/ml concentration with deionized water, 4 DEG C keep in Dark Place for subsequent use, use final concentration be 1 μ g/ml, 4 DEG C keep in Dark Place.Ultraviolet excitation, nucleus and chromosome are blue-fluorescence.
(4) phosphate buffer (PBS damping fluid)
NaCl 0.14mM 0.0082g/L
KCl 2.7mM 0.2013g/L
Na
2HPO
4 8.0mM 2.8651g/L
NaH
2PO
4 1.5mM 0.2041g/L
Add deionized water to 1L, use 0.1M NaH
2pO
4adjust pH to 7.0
(5) fixing agent 4% paraformaldehyde (paraformaldehyde in PBS buffer): purchased from Sigma (U.S.) company.Claim paraformaldehyde white powder 1g, measure 25ml PBS buffer in beaker, lid lid, is placed in fuming cupboard, and 90 DEG C are heated with stirring to dissolving (being Transparent color), are cooled to after room temperature, pour in brown bottle, and 4 DEG C of preservations are stand-by.Fixing agent 4% paraformaldehyde, is mainly cell killing, maintains the configuration state of cell in the time of live body.
(6) enzyme liquid (2.5%Cellulase cellulase+2.5%Pectolase pectase): purchased from Yakult Honsha (Japan) company.General preparation 5ml enzyme liquid, point another name 0.125g Cellulase and 0.125g Pectolase, in 5ml deionized water, after fully dissolving, be sub-packed in the centrifuge tube of 4 1.5ml, and-24 DEG C save backup.Main Function is removed cell membrane (cellulose and pectin).
(7) 1%Triton X-100 Triton X-100 (Qu Latong): purchased from Solution on Chemical Reagents in Shanghai company.One well matched 25ml, gets 0.25ml Triton X-100 and is added in 25ml PBS buffer, matching while using.This is a kind of surfactant of excellence, wetting and washing agent, and broken cell film punches on cell membrane, and antibody is entered.
(8) anti-fluorescent quenching mounting liquid (Antifade mounting medium): purchased from Shanghai Jie Mei genome company.
(9) nail polish market is purchased.
The problem that should note in experiment of the present invention:
(1) activity of antibody has determined result of use, and proper if antibody is preserved, most of antibody activity can maintain even several years several months.Therefore, the antibody of buying packing as early as possible, packing can farthest reduce the infringement of multigelation antagonist activity, has also reduced owing to repeatedly drawing the possibility of pollution that antibody causes from same pipe simultaneously.
(2) the paraformaldehyde reagent for fixed sample that the present invention uses belongs to hazardous chemical, please notes suitable protection.Paraformaldehyde is not soluble in cold water, will be placed in fuming cupboard when preparation, and 90 DEG C are heated with stirring to dissolving (being Transparent color), are cooled to after room temperature, pours in brown bottle, and 4 DEG C of preservations are stand-by.
The good effect that the present invention compared with prior art had is:
(1) the present invention detects the immunofluorescence method of vegetable cell kernel C23 protein positioning under heavy metal stress, can be to after heavy metal stress, and the variation of intracellular nucleic benevolence C23 protein is accurately located.Enzymolysis is wherein that this tests one of crucial step, and enzymolysis time is determined according to cell concrete condition.Divide equally and scatter if for example observe most cells in the time of microscopy, become the spherical bioplast of one deck, wherein the tenuigenin of a small amount of cell disintegrates, and only finishes enzymolysis when remaining core.
(2) the present invention reforms traditional flaking method.After enzymolysis, directly sample is made to suspending liquid state, then, dropped in equably on microslide, need not cover cover glass, for subsequent use after natural air drying.This method has deducted conventional compressing tablet middle cover cover plate and two steps of peel, has avoided the damage to cell in the operation of these two steps.
(3) detection technique of the present invention has the features, the particularly reform to conventional tabletting method such as specificity is high, experimental result is accurate, make operation steps more simple and easy to do, greatly improved the advantages such as observation of cell number.The method is for providing scientific basis to the impact of protein in cell under the environment stresses such as research heavy metal.Provide accurate detection method for inquiring into heavy metallic poison celelular mechanism, its method has broad application prospects.
The present invention, by immunofluorescence location technology, accurately locates the variation of the nucleolus C 23 protein after heavy metal stress.We utilize said method to observe under Al, Cd and Pb coerce, and the variation of nucleolus C 23 protein in onion and Vicia faba Root Tip Cells, has obtained good result.
Brief description of the drawings:
Fig. 1 comprises:
Diagram A1-A3 is the distribution without nucleolus C 23 protein in the cell of heavy metal stress.Wherein A1: fluorescent red-orange shows C23 albumen; A2: blue-fluorescence showed cell core (DNA); A3: composite diagram.
Diagram B-C is the location of nucleolus C 23 protein in cell in cell after heavy metal stress.C23 albumen in B, C showed cell core is excessive in tenuigenin under the coercing of heavy metal, and along with the rising of heavy metal concentration for the treatment of and the prolongation in processing time, C23 protein content increases gradually.Wherein:
B1: fluorescent red-orange shows C23 albumen; B2: blue-fluorescence showed cell core (DNA); B3: composite diagram;
C1: fluorescent red-orange shows C23 albumen; C2: blue-fluorescence showed cell core (DNA); C3: composite diagram;
Fig. 2 is that silver dyes (AgNO
3dyeing) experimental result picture.(Fig. 2 a) to be dark-brown without the entoblast of heavy metal stress.Cd
2+after processing, find some and nucleolar silver staining reacting phase with fine particle be distributed in that in tenuigenin, (Fig. 2 b).The quantity of these Ag-NORs is along with Cd
2+concentration raises and the prolongation in processing time and increase that (Fig. 2 c).
Embodiment
, and be not used in and also should be interpreted as by any way to the restriction of inventing in listed claim with helping understand the present invention below in conjunction with embodiment.Be illustrated especially, the present invention's various reagent used all has commercially available.
Embodiment 1
Material is cultivated taking broad bean as example: when broad bean master (side) root root is about 1.5cm, with the Al of 10 μ M, 50 μ M, 100 μ M
3+process 24h, 48h, 72h.
(1) cut heavy metal Bean Plant after treatment apical meristem cell, in 4% paraformaldehyde, fix 2h; Wash through PBS damping fluid;
(2) adopt 2.5% cellulase+2.5% pectase enzyme liquid in 37 DEG C of incubator enzymolysis; Compressing tablet after the washing of PBS damping fluid;
(3) the Bean Plant tip of a root is gone in centrifuge tube, drip PBS damping fluid, hand concussion, to most cell free states, is drawn about 0.1ml sample liquid with dropper, is evenly applied on microslide and is dispersed into individual cells, for subsequent use after natural air drying after mark;
(4) microslide (3) being obtained is placed on 1%TritonX-100 and soaks 15 minutes; Wash through PBS damping fluid;
(5) on the slide glass obtaining in (4), splash into the primary antibodie that 15 μ l prepare, covered, 37 DEG C of incubation 1h; Wash through PBS damping fluid again;
(6) what on the slide glass again (5) being obtained, splash into that 15 μ l prepare is two anti-, covered, 37 DEG C of incubation 45min-1h; Wash through PBS damping fluid;
(7) 15 μ l DAPI, covered will be dripped again on the microslide of (6); Wash through PBS damping fluid;
(8) blot unnecessary PBS with filter paper, drip 5 μ l anti-quencher on microslide material, covered, seals cover glass surrounding with nail polish, after 1h at fluorescence microscopy Microscopic observation.
(9) fluorescence microscope and image analysis processing: (Japan) HB-10101AF of NIKON company for film-making type fluorescence microscope, Pixera Pro 600CL CCD digital photographing.Image acquisition is 1392 × 1040 pixels, and the post-processed of image is utilized Photoshop 7.0 software typesettings.C23 albumen is by TRITC mark, and blue light 450～490nm excites, the fluorescence that takes on a red color (seeing A1 in Fig. 1, B1, C1), and nucleus is dyeed by DAPI, and ultraviolet light 355～425nm excites, and is blue-fluorescence (seeing A2 in Fig. 1, B2, C2).A3 in Fig. 1, B3, C3 is composite diagram.
Embodiment 2
Material is cultivated taking garlic as example, when garlic adventitious root root is about 1.5cm, with the Pb of 10 μ M, 50 μ M, 100 μ M
2+process 24h, 48h, 72h.
Experimental procedure:
(1) cut the about 2mm of the heavy metal tip of a root after treatment, in 4% paraformaldehyde, fix 2h.
(2) in PBS damping fluid (pH7.0), wash (each 10 minutes) 3 times.
(3) 2.5% cellulase+2.5% pectases, 37 DEG C of incubator enzymolysis, microscopy at any time in enzymolysis process, finishes enzymolysis until there is a large amount of spherical bioplasts to produce.
(4) PBS damping fluid is washed (each 10 minutes) 3 times.
(5) tip of a root is gone in the centrifuge tube of 0.5ml, drip PBS damping fluid a small amount of (suitable sample how many and determine), lid centrifuge tube lid, hand concussion is most cell free states extremely.Draw about 0.2ml sample liquid with dropper, be evenly applied on slide glass and be dispersed into individual cells, do not cover cover glass, the n.s. face mark with marking pen at slide glass, for subsequent use after natural air drying.
(6) section that will mark is placed on extracting 20min in 1%Triton X-100.
(7) PBS damping fluid is washed (each 10 minutes) 3 times.
(8) blot unnecessary PBS damping fluid with filter paper, use through 37 DEG C of the primary antibodies (mouse-anti C23 monoclonal antibody, working concentration 1: 150) of PBS dilution and hatch 1h or 4 DEG C of overnight incubation.
(9) in PBS damping fluid, wash (each 10 minutes) 3 times.
(10) blot unnecessary PBS damping fluid with filter paper, drip that (two anti-(mountain sheep anti-mouse igg, the working concentrations 1: 50) that 20 μ l) prepare are to the sample place on microslide, covered, 37 DEG C of incubation 45min-1h.
(11) in PBS damping fluid, wash (each 10 minutes) 3 times.
(12) DAPI dyeing: blot unnecessary PBS damping fluid with filter paper, drip (l) DAPI of 20 μ, covered.
(13) in PBS damping fluid, wash (each 10 minutes) 3 times.
(14) blot unnecessary PBS with filter paper, drip that (l) anti-quencher is on microslide material for 10 μ, and covered, seals cover glass surrounding with nail polish, can be at fluorescence microscopy Microscopic observation after 1h.
(15) fluorescence microscope and image analysis processing: (Japan) HB-10101AF of NIKON company for film-making type fluorescence microscope, Pixera Pro 600CL CCD digital photographing.Image acquisition is 1392 × 1040 pixels, and the post-processed of image is utilized Photoshop 7.0 software typesettings.C23 albumen is by TRITC mark, and blue light 450～490nm excites, the fluorescence that takes on a red color (seeing A1 in Fig. 1, B1, C1), and nucleus is dyeed by DAPI, and ultraviolet light 355～425nm excites, and is blue-fluorescence (seeing A2 in Fig. 1, B2, C2).A3 in Fig. 1, B3, C3 is composite diagram.
Embodiment 3
The variation of nucleolus C 23 protein in Onion root-tip cell
Material is cultivated taking onion as example: when onion adventitious root root is about 1.5cm, with the Cd of 10 μ M, 50 μ M, 100 μ M
2+process 24h, 48h, 72h.
Experimental procedure:
(1) cut the about 2mm of the heavy metal tip of a root after treatment, in 4% paraformaldehyde, fix 2h.
(2) in PBS damping fluid (pH7.0), wash (each 15 minutes) 3 times.
(3) 2.5% cellulase+2.5% pectases, 37 DEG C of incubator enzymolysis, microscopy at any time in enzymolysis process, finishes enzymolysis until there is a large amount of spherical bioplasts to produce.
(4) PBS damping fluid is washed (each 15 minutes) 3 times.
(5) tip of a root is gone in the centrifuge tube of 0.5ml, drip PBS damping fluid a small amount of (suitable sample how many and determine), lid centrifuge tube lid, hand concussion is most cell free states extremely.Draw about 0.1ml sample liquid with dropper, be evenly applied on slide glass and be dispersed into individual cells, do not cover cover glass, the n.s. face mark with marking pen at slide glass, for subsequent use after natural air drying.
(6) section that will mark is placed on extracting 20min in 1%Triton X-100.
(7) PBS damping fluid is washed (each 15 minutes) 3 times.
(8) blot unnecessary PBS damping fluid with filter paper, use through 37 DEG C of the primary antibodies (mouse-anti C23 monoclonal antibody, working concentration 1: 150) of PBS dilution and hatch 1h or 4 DEG C of overnight incubation.
(9) in PBS damping fluid, wash (each 10 minutes) 3 times.
(10) blot unnecessary PBS damping fluid with filter paper, drip that (two anti-(mountain sheep anti-mouse igg, the working concentrations 1: 50) that 20 μ l) prepare are to the sample place on microslide, covered, 37 DEG C of incubation 45min.
(11) in PBS damping fluid, wash (each 10 minutes) 3 times.
(12) DAPI dyeing: blot unnecessary PBS damping fluid with filter paper, drip that (20 μ are DAPI covered l).
(13) in PBS damping fluid, wash (each 10 minutes) 3 times.
(14) blot unnecessary PBS with filter paper, drip that (l) anti-quencher is on microslide material for 10 μ, and covered, seals cover glass surrounding with nail polish, can be at fluorescence microscopy Microscopic observation after 1h.
(15) fluorescence microscope and image analysis processing: (Japan) HB-10101AF of NIKON company for film-making type fluorescence microscope, Pixera Pro 600CL CCD digital photographing.Image acquisition is 1392 × 1040 pixels, and the post-processed of image is utilized Photoshop 7.0 software typesettings.The free TRITC mark of C23 egg, blue light 450～490nm excites, the fluorescence that takes on a red color (seeing A1 in Fig. 1, B1, C1), nucleus is dyeed by DAPI, and ultraviolet light 355～425nm excites, and is blue-fluorescence (seeing A2 in Fig. 1, B2, C2).A3 in Fig. 1, B3, C3 is composite diagram.
Embodiment 4
The comparison test of silver dyeing technique (Ag-NOR staining technique) and the technology of the present invention:
Silver dyes principle: Ag-NOR staining technique can be specifically with cell in NOR (NOR) dyeing.In the time being positioned at the gene (rDNA) of 18S herein, 28S rRNA and thering is transcriptional activity, apply silver-colored dyeing technique and can make NOR specificity painted (brown color).Now further proving that it is not rDNA that silver dyes material, is not also rRNA, may be the special protein of NOR, transcribes with rRNA the acidic protein being associated.
Material is cultivated taking broad bean as example, when Roots of Vicia Faba is about 1.5cm, with the Cd of 10 μ M, 50 μ M, 100 μ M
2+process 24h, 48h, 72h.
Experimental procedure:
(1) cut the about 2mm of the heavy metal tip of a root after treatment, in immobile liquid (alcohol: acetic acid=3: fix 1h 2).
(2) in deionized water, wash (each 10 minutes) 3 times.
(3) be placed in 60 DEG C of thermostat water baths, with hydrolyzate (1NHCl: alcohol: acetic acid=5: 3: 2) hydrolysis 9min.
(4) in deionized water, wash (each 10 minutes) 3 times.
(5) 45% acetic acid compressing tablet for the tip of a root, dried overnight under room temperature, peel.
(6) on dry sliced, add 1 2% gelatin solution and 2 50%AgNO
3aqueous solution, mixes, and with cover plate, under room temperature, contaminates.
(7) in the time that kernel is brown color, tenuigenin and is light yellow, with distilled water by clean dyeing liquor drip washing.
(8) redye at 0.001% aqueous solution of methylene blue, be green to chromatin.
(9) with distilled water by clean dyeing liquor drip washing, dry under room temperature.
(10) optical resin mounting, micro-Microscopic observation.
(11) microscopic examination and image analysis processing: film-making is observed with VANOX-AHB type Olympus microscope (Japan), Pixera Pro 600CL CCD digital photographing.Image acquisition is 1392 × 1040 pixels, and the post-processed of image is utilized Photoshop 7.0 software typesettings.Through AgNO
3dyeing, kernel is brown color, tenuigenin is light yellow, and chromosome is dyeed by methylene blue, is green (seeing Fig. 2).
Silver dyes experimental result: (Fig. 2 a) to be dark-brown without the entoblast of heavy metal stress.Cd
2+after processing, find some and nucleolar silver staining reacting phase with fine particle be distributed in that in tenuigenin, (Fig. 2 b).The quantity of these Ag-NORs is along with Cd
2+concentration raises and the prolongation in processing time and increase that (Fig. 2 c).
Adopt method of the present invention to measure and result: to be positioned at kernel (Figure 1A) without nucleolus C 23 protein in the cell of heavy metal stress.After heavy metal stress, in cell, nucleolus C 23 albumen is distributed in caryoplasm (Figure 1B), and further excessive in tenuigenin (Fig. 1 C).
The comparison of two kinds of experimental techniques:
Utilize the impact of silver-colored dyeing technique research counterweight metal pair vegetable cell kernel structure, there is certain scientific value.But, silver dyeing technique can only be identified after heavy metal stress, be in kernel, to have a liking for silver-colored acidic protein from the excessive Ag-NOR to tenuigenin of nucleus, can not identify it is the impact which kind nucleolin is subject to heavy metal stress, further investigation heavy metallic poison mechanism is had to certain limitation.
The present invention, by immunofluorescence location technology, accurately locates the variation of the nucleolus C 23 protein after heavy metal stress, has the features such as specificity is high, experimental result is accurate.Detection technique of the present invention can further deeply detect the impact that heavy metal causes concrete nucleolar structure composition.The method, for providing scientific basis to the impact of protein in cell under the environment stresses such as research heavy metal, provides accurate detection method for inquiring into heavy metallic poison celelular mechanism, and its method has broad application prospects.
After the preferred embodiment describing in detail, being familiar with this technology personage can be well understood to, can carry out various variations and amendment not departing under above-mentioned claim and spirit, any simple modification, equivalent variations and modification that all foundations technical spirit of the present invention is done above embodiment, all belong to the scope of technical solution of the present invention.And the present invention is not also subject to the restriction of example embodiment in instructions.
Claims (6)
1. an immunofluorescence method that detects vegetable cell kernel C23 protein positioning under heavy metal stress, is characterized in that, is undertaken by following step:
(1) cut heavy metal plant root tip meristematic cell after treatment, in 4% paraformaldehyde, fix 1-2h; Wash through PBS damping fluid;
(2) adopt 2.5% cellulase+2.5% pectase enzyme liquid in 37 DEG C of incubator enzymolysis, microscopy at any time in enzymolysis process, finishes enzymolysis until there is a large amount of spherical bioplasts to produce, compressing tablet after the washing of PBS damping fluid;
(3) plant root tip is gone in centrifuge tube, drip PBS damping fluid, hand concussion, to most cell free states, is drawn 0.1-0.2ml sample liquid with dropper, is evenly applied on microslide and is dispersed into individual cells, for subsequent use after natural air drying after mark;
(4) microslide (3) being obtained is placed on 1%TritonX-100 and soaks 15-20 minute; Wash through PBS damping fluid;
(5) on the slide glass obtaining in (4), splash into the primary antibodie that 15-20 μ l prepares, covered, 37 DEG C of incubation 1h-1.5h; Wash through PBS damping fluid again; Described primary antibodie is: mouse-anti C23 monoclonal antibody, and its concentration is 0.2mg/ml;
(6) what on the slide glass again (5) being obtained, splash into that 15-20 μ l prepares is two anti-, covered, 37 DEG C of incubation 45min-1h; Wash through PBS damping fluid; Described two resist and are: TRITC-mountain its concentration of sheep anti-mouse igg is 1.5mg/ml;
(7) 15-20 μ l DAPI, covered will be dripped again on the microslide of (6); Wash through PBS damping fluid;
(8) blot unnecessary PBS with filter paper, drip 5-10 μ l anti-quencher on microslide material, covered, seals cover glass surrounding with nail polish, after 1-2h at fluorescence microscopy Microscopic observation; Wherein the concentration of DAPI is 1 μ g/ml.
2. detection method claimed in claim 1, wherein the enzymolysis time used refers in step (2): divide equally and scatter if observe most cells in the time of microscopy, become the spherical bioplast of one deck, wherein the tenuigenin of a small amount of cell disintegrates, only when remaining nucleus, finish enzymolysis.
3. detection method claimed in claim 1, wherein the compressing tablet described in step (2) refers to: after enzymolysis, directly sample is made to suspending liquid state, then, dropped in equably on microslide, do not cover cover glass, for subsequent use after natural air drying.
4. detection method claimed in claim 1, wherein said PBS damping fluid is washed and is referred to and adopt PBS damping fluid to wash 3 times, each 10-15 minute.
5. detection method claimed in claim 1, heavy metal is wherein Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb or Zn.
6. detection method claimed in claim 1, wherein said vegetable cell comprises broad bean, onion, garlic or corn.
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| CN101294903A (en) * | 2008-06-03 | 2008-10-29 | 武汉大学 | Method for detecting identical antigen expression on identical sample |
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