CN103777001A - Hypersensitive immune electron microscope marking method - Google Patents
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- CN103777001A CN103777001A CN201410036229.0A CN201410036229A CN103777001A CN 103777001 A CN103777001 A CN 103777001A CN 201410036229 A CN201410036229 A CN 201410036229A CN 103777001 A CN103777001 A CN 103777001A
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Abstract
The invention discloses a hypersensitive immune electron microscope marking method. The method comprises the following steps: fixing and dehydrating a to-be-tested antigen of a biological sample by using a glutaraldehyde solution of which the percent by volume is 0.1-2.5 percent and a paraformaldehyde solution of which the percent by mass is 1.5-5 percent, subsequently embedding the to-be-tested antigen in hydrophilic resin, slicing the embedded to-be-tested antigen into ultrathin slices, respectively marking the to-tested antigen of the biological sample by using specific first antibodies, then marking the first antibodies by using second antibodies on which ultra-micro gold particles are adsorbed, forming large-diameter silver particles around the ultra-micro gold particles by using a silver reinforcing and amplifying solvent, and then sensitively detecting the epi-position of the to-be-tested antigen in the biological sample under an electron microscope. The hypersensitive immune electron microscope marking method disclosed by the invention has the advantages that the sensitivity is high, positive signal particles are relatively uniform, convenience is provided for study and judgment, and simultaneously the antigen inside the biological sample can be detected. The hypersensitive immune electron microscope marking method disclosed by the invention is suitable for detecting trace of antigen inside a biomedical sample.
Description
Technical field
The invention belongs to modern biomedical technical field, particularly a kind of super quick immuno-electron microscope labeling method.
Background technology
Immuno-electron microscope is to utilize immunohistochemistry technology and electron microscopy in ultrastructure level, to observe the pinpoint method of determined antigen in biomedical samples.What immunoelectronmicroscopy was in one's early years used is enzyme linked immunosorbent assay, and what present immunoelectronmicroscopy was often used is the anti-method of gold mark two.Two is anti-also referred to as second antibody, a kind of protein, it is anti-that gold grain that can be different from diameter forms gold mark two by electrostatic interaction, if this gold mark two is anti-and an anti-binding, primary antibodie is its corresponding antigen generation specificity coupled action again, under Electronic Speculum, will form the dense granule that represents corresponding antigens epi-position.Use at present gold mark two anti-immunoelectronmicroscopies to be divided into before embedding the large class of immuno-electron microscope two after immuno-electron microscope and embedding.
Immuno-electron microscope before embedding, as the term suggests be exactly just to carry out immune labeled to biological sample to be measured through before the embedding program of electron microscopy.Generally speaking, the diameter of common antibody is 10nm, when with gold grain generation electrostatical binding, the diameter of gold grain is less, be attached to antibody gold grain around also just more, in mark, elution process, be also more not easy to make gold grain to lose, the particle of the representative determined antigen epi-position showing under Electronic Speculum is also just more.That is to say, and the gold grain diameter of two anti-generation electrostatical bindings is less, in the time detecting determined antigen, susceptibility is higher.If but diameter is too little, if gold grain diameter is when the 0.8nm-1.4nm, even be also not easy to observe gold grain under the high power pattern of Electronic Speculum, therefore, before embedding, immunoelectronmicroscopy often needs to utilize silver to strengthen reagent, make position take gold grain as the core silver-colored particle of diameter 5-50nm in enrichment again, thereby indirectly demonstrate the epi-position of biological sample determined antigen.
The advantage of immuno-electron microscope before embedding: the susceptibility of detection is high.Because before embedding, immuno-electron microscope uses is that diameter is the ultra micro gold grain of 0.8nm-1.4nm, two of diameter 10-15nm anti-is easy to so tiny ultra micro gold grain of absorption, and can resist strict rinsing condition.After silver is strengthened step, can show the accurate epi-position of determined antigen very sensitively, and the incubation time of can be as required strengthening reagent by extending silver control the silver-colored particle diameter of ultra micro gold grain enrichment around, make it reach the susceptibility of amplifying effect doubly of 5-50 and do not affect mark.
The shortcoming of immuno-electron microscope before embedding.First sample (as embodiment 1) that, can only mark sample shallow-layer.It is that the gold mark two of 0.8nm-1.4nm is anti-that absorption has diameter, even in the situation that adding penetration agent, its penetration capacity is very limited, generally all can be lower than 10 μ m, antigen that can not mark biological sample deep layer, can cut into slices by vibration as brain sheet for the biological sample of bulk, if very little biological sample, as the organ such as embryo, brain of fruit bat, is just difficult to reach mark effect.Secondly, in order to increase the degree of depth of testing sample, before embedding, in immuno-electron microscope running program, tend to use certain density non-ionic detergent Tween-20 or surfactant Triton X-100, this two classes chemical substance tends to cause biological sample ultrastructure damage or lose, thereby may affect the detection of determined antigen.Again, before embedding, immuno-electron microscope is in the time using silver to strengthen amplification procedure, because cell interior structure and the component distribution height of biological sample are inhomogeneous, silver particle is in the time that enrichment around gold grain forms dense-core, it is inconsistent that it penetrates distance and path that biological sample to be measured enters, by causing the last silver-colored granular core diameter forming to differ (as embodiment 1), if these diameters differ larger, the judgement to detection architecture will be disturbed, and quantitative test can not be done.
Immuno-electron microscope after embedding, as the term suggests be exactly just to carry out immune labeled to biological sample to be measured after the process embedding program of electron microscopy.
The advantage of immuno-electron microscope after embedding.First determined antigen that, can detection of biological sample interior.Secondly, after embedding, immuno-electron microscope does not use penetration agent, can make the ultrastructure of biological sample to be measured and determined antigen be preserved preferably; Again, according to the quantity of gold grain, can make semi-quantitative analysis to testing sample.
The shortcoming of immuno-electron microscope after embedding.Because the gold that immuno-electron microscope after embedding uses is marked two anti-gold grain diameters and reached 5-50nm, and the two anti-diameters of being combined with gold grain are approximately 10-15nm, with it the gold grain quantity of combination than embedding before the ultra micro gold grain quantity of 0.8-1.4nm that uses of immuno-electron microscope much lower, and gold grain is easily lost in mark, elution process, therefore the susceptibility of immuno-electron microscope mark low (as embodiment 2) after embedding, and gold grain is larger, susceptibility is lower.On the other hand, the ultra-thin section general thickness of biological sample is 100nm left and right, if utilize the gold mark two that diameter is larger anti-, its ability that penetrates sample also can be affected.Therefore,, with the determined antigen of immunoelectronmicroscopy detection of biological sample after embedding, susceptibility will reduce.
Summary of the invention
Goal of the invention: the problem and shortage existing for above-mentioned prior art, the present invention fully combines before embedding the advantage of immuno-electron microscope after immuno-electron microscope and embedding, and a kind of immuno-electron microscope labeling method of hypersensitization is provided.
Technical scheme: in order to solve the problems of the technologies described above, technical scheme provided by the invention is as follows: a kind of super quick immuno-electron microscope labeling method, comprise the following steps: the glutaraldehyde solution that is 0.1-2.5% biological sample determined antigen percent by volume and mass percent are after 1.5-5% paraformaldehyde solution is fixed, after dehydration procedure, through hydrophilic resin embedding, be cut into after ultra-thin section, use respectively specific first antibody mark biological sample determined antigen, again to adsorb the second antibody mark first antibody of ultra micro gold grain, then utilize silver to strengthen amplifying reagent and around ultra micro gold grain, form the silver-colored particle that diameter is larger, then under Electronic Speculum, can detect sensitively the epi-position of determined antigen in biological sample.
Wherein, above-mentioned hydrophilic resin is K4M or L.R.White.
Wherein, above-mentioned ultra-thin section thickness is 60-100nm.
Wherein, ultra micro gold grain diameter is 0.8nm-1.4nm.
Concrete steps of the present invention are as follows: utilize immuno-electron microscope program after embedding, by biological sample to be measured through being dissolved in the phosphate buffer of 0.1M or the 1.5-5% paraformaldehyde solution of sodium cacodylate buffer liquid and 0.1-2.5% glutaraldehyde two fixing (PH7.0-7.6), after the phosphate rinsing and dehydration of 0.1M, be embedded in hydrophilic resin K4M or L.R.White, and make sample in resin, aggregate into hard bulk; In ultramicrotome, be cut into the ultra-thin section of 60-100nm being embedded in biological sample to be measured in hydrophilic resin K4M or L.R.White, and be collected in golden net or nickel screen; The golden net or the nickel screen that are loaded with biological sample to be measured are hatched after 5 minutes with deionized water, again to seal in the confining liquid that contains BSA 30 minutes, then hatch 30 minutes with first antibody, then wash three times with the confining liquid that contains BSA, to wash away the first antibody that there is no specific binding; By the second antibody mark first antibody of the ultra micro gold mark of 0.8nm or 1.4nm 30 minutes, then with the confining liquid washing that contains BSA three times, to wash away the second antibody that there is no specific binding; Strengthen reagent with silver the ultra micro gold grain of the second antibody in nickel screen is amplified, the time of amplification is controlled at 10-30 minute as required, and the time is longer, and the silver-colored particle crystallization forming on ultra micro gold grain surface is larger, more easily observes.By the nickel screen process 0.1MPBS(PH=7.4 that is loaded with biological sample to be measured that strengthens through silver amplifying) wash three times, fixing after 1% glutaraldehyde, then after conventional lead citrate and uranium acetate dyeing, under transmission electron microscope, observe, take pictures.
Beneficial effect: compared with prior art, before the present invention has overcome embedding, immunoelectronmicroscopy penetrates the weak drawback of biological sample ability, retain its sensitive high advantage, after having overcome embedding, immunoelectronmicroscopy is sensitive low, but penetrates the advantage of biological sample inside (ultra-thin section that after embedding, immunoelectronmicroscopy is used is 60-100nm) without antibody.The present invention is applicable to detecting the detection of the trace antigen that is positioned at biomedical sample inside, and the positive signal occurring is relatively even, is convenient to judgement.Utilize method of the present invention, take synaptic vesicle labeled molecule synaptophysin (Synapsin) mark synaptic vesicle as example, utilizing diameter is that the gold of 1.4 nanometers is marked two anti-mark synaptophysins, strengthening reagent with silver again amplifies, two anti-mark synaptophysins are directly marked in contrast take diameter as the gold of 15 nanometers, its sensitivity can improve 10 times above (seeing specific embodiments 2), and positive signal is relatively even, is convenient to judgement.If adopt diameter less, mark two anti-mark corresponding antigens as the gold of 0.8 nanometer, its sensitivity also will significantly increase.
Accompanying drawing explanation
Figure 1A demonstration utilizes the fruit bat neuromuscular junction of the front immuno-electron microscope program of embedding with the DLG antibody labeling of fruit bat, because shallow-layer is being organized in the neuromuscular junction of fruit bat periphery, mark result can be light Microscopic observation (sphere shown in white box be the drosophila larvae neuromuscular junction of mark);
The expression sites of the fruit bat neuromuscular junction that Figure 1B is presented at the DLG antibody labeling with fruit bat of boxed area in Figure 1A under Electronic Speculum, DLG expresses in postsynaptic broad regions, but not expresses at postsynaptic membrane, with consistent described in forefathers.The positive detection rate of DLG antibody under Electronic Speculum is very high, positive signal is very intensive, and black granule shows positive signal, and black arrow is shown the cell membrane of the muscle cell communicating with external environment, show by immune programme for children before embedding, antibody can only be through the region of 10 microns of left and right;
Stain in Fig. 1 C is all positive signal, but the diameter that shown in large, medium and small arrow, positive signal occurs differs huge;
Fig. 1 D shows the central nervous system of fruit bat, and what white line showed is a tangent plane of ventral nerve corb;
Fig. 1 E shows the section that Fig. 1 D takes along white line, and under Electronic Speculum, the synaptic vesicle of fruit bat is positioned at the middle section of section, utilizes immuno-electron microscope program before traditional embedding can not make signal on the middle section mark of this section; Reach the object of mark, at present only by means of immuno-electron microscope program after embedding (scale of noting E figure is 5 microns);
Fig. 2 A shows normal synaptic knob and synaptic vesicle structure and the position under Electronic Speculum, synaptic knob glomeration, and synaptic vesicle is distributed in synaptic knob periphery;
Fig. 2 B shows the signal of the synaptophysin Synapsin of mark of the present invention, represent the distribution pattern of synaptic vesicle in synaptic knob, result demonstration, positive signal is distributed in synaptic knob periphery, and the distribution pattern of the synaptic vesicle that this distribution pattern and Fig. 2 A show on synaptic knob is in full accord;
Fig. 2 C shows the signal of the synaptophysin Synapsin of immuno-electron microscope program mark after traditional embedding, and these signals are very sparse, even on a lot of samples, is not observing.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.
DLG in the present invention, synapsin primary antibodie and 15nm gold mark two are anti-purchased from Sigma company, and the anti-and silver of the mouse source gold mark two of 1.4nm is strengthened reagent purchased from Nanogold molecular probe company, and the mouse source gold mark two of 0.8nm is anti-purchased from AURION company.
Embodiment 1
DLG expresses in the postsynaptic half cynapse reticular tissue of drosophila larvae neuromuscular junction, and in neurobiological study, DLG shows one of the conventional labeled molecule in drosophila larvae neuromuscular junction postsynaptic region (not being at postsynaptic membrane).Because drosophila larvae neuromuscular junction is positioned at the top layer of muscle, utilize the front immuno-electron microscope program of embedding, take the anti-DLG mouse source monoclonal antibody of fruit bat as primary antibodie (working concentration 1:20), again with the two anti-processing (working concentration 1:50) with 1.4 nanogold particle marks of anti-mouse, then carry out silver and strengthen amplifying (amplification condition be at 25 ℃ 10 minutes), the positive signal of a large amount of DLG can be detected in postsynaptic region, can't detect signal (Figure 1B-C) at postsynaptic membrane, this and people's cognitive consonance, illustrates that present case is out of question in operation.But the diameter of these signals differs larger (Fig. 1 C), can bring the possibility of erroneous judgement to detection.And immune programme for children can only detect the sample that is positioned at 10 microns of left and right of sample surfaces before embedding, if in the face of being positioned at the antigen of organizing deep layer, as shown in Fig. 1 D-E, utilize the front immuno-electron microscope program of embedding just helpless.
Embodiment 2
Synaptophysin (Synapsin) is a kind of albumen being positioned on synaptic vesicle, is one of mark albumen of synaptic vesicle.In the process of specific embodiments of the invention 2, immune labeled experimental arrangement and content of the present invention used are just the same.Fixing agent used is the 0.1M PBS solution (PH=7.4) of 0.5% glutaraldehyde+4% paraformaldehyde, and the ventral nerve corb that animal tissue used is fruit bat, is intensively distributed with synaptic knob in it, and synaptic vesicle is looped around (Fig. 2 A) around synaptic knob.Primary antibodie used is the mouse source monoclonal antibody synaptophysin of specific recognition fruit bat synaptic vesicle, and working concentration is 1:20.Used two anti-have two kinds, and a kind of is that diameter is gold mark mouse two anti-of 1.4 nanometers, after mark completes, strengthen reagent amplify gold grain diameter with silver, and the condition that silver strengthens amplifying is at 25 ℃ 10 minutes; Another kind of two anti-be that diameter is that the gold of 15 nanometers is marked the two anti-of anti-mouse, directly mark first antibody; The working concentration of two kinds of second antibody is 1:50.Experiment shows, under duplicate experiment condition, utilize the solution of the present invention, mark two anti-marks take diameter as the gold of 1.4 nanometers, strengthening reagent amplifying signal with silver again, the frequency (Fig. 2 B) of the appearance of synaptophysin Synapsin is far longer than the frequency (Fig. 2 C) directly occurring with the direct mark post-synapse element of 15nm Synapsin, in two typical samples selecting in experiment, 26 positive signal (Fig. 2 B are observed at most with program of the present invention, shown in arrow), and can only observe at most two positive signal or even not observe positive signal (Fig. 2 C with immune programme for children after traditional embedding, shown in arrow).Compared with immune programme for children before traditional embedding (Fig. 1 C, shown in arrow), there is relatively evenly (Fig. 2 B, shown in arrow) of positive signal diameter in the present invention, is convenient to judgement.
Embodiment 3
By biological sample to be measured through being dissolved in 2% paraformaldehyde of phosphate buffer of 0.1M and 0.1% glutaraldehyde two fixing (PH7.0-7.6), after the phosphate rinsing and dehydration of 0.1M, be embedded in hydrophilic resin K4M; In ultramicrotome, be cut into the ultra-thin section of 60nm being embedded in biological sample to be measured in hydrophilic resin K4M, and be collected in golden net or nickel screen; The golden net or the nickel screen that are loaded with biological sample to be measured are hatched after 5 minutes with deionized water, again to seal in the confining liquid that contains BSA 30 minutes, then hatch 30 minutes with first antibody, then wash three times with the confining liquid that contains BSA, to wash away the first antibody that there is no specific binding; By the second antibody mark first antibody of 0.8nm colloid gold label 30 minutes, then with the confining liquid washing that contains BSA three times, to wash away the second antibody that there is no specific binding; Strengthening kit with silver amplifies the ultra micro gold grain of the second antibody in nickel screen, the time of amplifying is controlled at 30 minutes as required, by the nickel screen process 0.1MPBS(PH=7.4 that is loaded with biological sample to be measured that strengthens through silver amplifying) wash three times, fixing after 1% glutaraldehyde, after conventional lead citrate and uranium acetate dyeing, under transmission electron microscope, observe, take pictures again.Animal tissue used is the ventral nerve corb of fruit bat, is intensively distributed with synaptic knob in it, and synaptic vesicle is looped around around synaptic knob.Primary antibodie used is the mouse source monoclonal antibody synaptophysin of specific recognition fruit bat synaptic vesicle, and working concentration is 1:20.Used two anti-have two kinds, and a kind of is that diameter is gold mark mouse two anti-of 0.8nm nanometer, after mark completes, strengthen kit amplify gold grain diameter with silver, and the condition that silver strengthens amplifying is at 25 ℃ 10 minutes; Another kind of two anti-be that diameter is that the gold of 15 nanometers is marked the two anti-of anti-mouse, directly mark first antibody; The working concentration of two kinds of second antibody is 1:50.Experiment shows, under duplicate experiment condition, utilize the solution of the present invention, mark two anti-marks take diameter as the gold of 0.8nm nanometer, strengthening amplifying signal with silver, the frequency of the appearance of synaptophysin Synapsin is far longer than the frequency directly occurring with the direct mark post-synapse element of 15nm Synapsin, in two typical samples selecting in experiment, observe at most 26 positive signal with program of the present invention, and can only observe at most two positives or even not observe positive signal with immune programme for children after traditional embedding.
Embodiment 4
By biological sample to be measured through being dissolved in 5% paraformaldehyde of sodium cacodylate buffer liquid of 0.1M and 0.25% glutaraldehyde two fixing (PH7.0-7.6), after the phosphate rinsing and dehydration of 0.1M, be embedded in hydrophilic resin L.R.White; In ultramicrotome, be cut into the ultra-thin section of 10nm being embedded in biological sample to be measured in hydrophilic resin L.R.White, and be collected in golden net or nickel screen; The golden net or the nickel screen that are loaded with biological sample to be measured are hatched after 5 minutes with deionized water, again to seal in the confining liquid that contains BSA 30 minutes, then hatch 30 minutes with first antibody, then wash three times with the confining liquid that contains BSA, to wash away the first antibody that there is no specific binding; By the second antibody mark first antibody of 0.8nm colloid gold label 30 minutes, then with the confining liquid washing that contains BSA three times, to wash away the second antibody that there is no specific binding; Strengthening kit with silver amplifies the ultra micro gold grain of the second antibody in nickel screen, the time of amplifying is controlled at 30 minutes as required, by the nickel screen process 0.1MPBS(PH=7.4 that is loaded with biological sample to be measured that strengthens through silver amplifying) wash three times, fixing after 1% glutaraldehyde, after conventional lead citrate and uranium acetate dyeing, under transmission electron microscope, observe, take pictures again.Animal tissue used is the ventral nerve corb of fruit bat, is intensively distributed with synaptic knob in it, and synaptic vesicle is looped around around synaptic knob.Primary antibodie used is the mouse source monoclonal antibody synaptophysin of specific recognition fruit bat synaptic vesicle, and working concentration is 1:20.Used two anti-have two kinds, and a kind of is that diameter is gold mark mouse two anti-of 1.4nm nanometer, after mark completes, strengthen kit amplify gold grain diameter with silver, and the condition that silver strengthens amplifying is at 25 ℃ 10 minutes; Another kind of two anti-be that diameter is that the gold of 15 nanometers is marked the two anti-of anti-mouse, directly mark first antibody; The working concentration of two kinds of second antibody is 1:50.Experiment shows, under duplicate experiment condition, utilize the solution of the present invention, mark two anti-marks take diameter as the gold of 0.8nm nanometer, strengthening amplifying signal with silver, the frequency of the appearance of synaptophysin Synapsin is far longer than the frequency directly occurring with the direct mark post-synapse element of 15nm Synapsin, in two typical samples selecting in experiment, observe at most 35 positive signal with program of the present invention, and can only observe at most two positives or even not observe positive signal with immune programme for children after traditional embedding.
Claims (4)
1. a super quick immuno-electron microscope labeling method, it is characterized in that, comprise the following steps: the glutaraldehyde solution that is 0.1-2.5% biological sample determined antigen percent by volume and mass percent are after 1.5-5% paraformaldehyde solution is fixed, after dehydration procedure, through hydrophilic resin embedding, be cut into after ultra-thin section, use respectively specific first antibody mark biological sample determined antigen, again to adsorb the second antibody mark first antibody of ultra micro gold grain, then utilize silver to strengthen amplifying reagent and around ultra micro gold grain, form the silver-colored particle that diameter is larger, then under Electronic Speculum, can detect sensitively the epi-position of determined antigen in biological sample.
2. a kind of super quick immuno-electron microscope labeling method according to claim 1, is characterized in that, described hydrophilic resin is K4M or L.R.White.
3. a kind of super quick immuno-electron microscope labeling method according to claim 1, is characterized in that, described ultra-thin section thickness is 60-100nm.
4. a kind of super quick immuno-electron microscope labeling method according to claim 1, is characterized in that, ultra micro gold grain diameter is 0.8nm-1.4nm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111257361A (en) * | 2020-03-13 | 2020-06-09 | 苏州智享众创孵化管理有限公司 | Electronic microscope quantitative detection method for virus particles by taking nano particles as reference substance |
| CN113405869A (en) * | 2021-06-09 | 2021-09-17 | 宁波大学 | Preparation method of protozoan cyst transmission electron microscope sample |
| CN113418766A (en) * | 2021-06-24 | 2021-09-21 | 中国科学院华南植物园 | Double-fixation embedding sample preparation method after whole immunization |
-
2014
- 2014-01-24 CN CN201410036229.0A patent/CN103777001A/en active Pending
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111257361A (en) * | 2020-03-13 | 2020-06-09 | 苏州智享众创孵化管理有限公司 | Electronic microscope quantitative detection method for virus particles by taking nano particles as reference substance |
| CN113405869A (en) * | 2021-06-09 | 2021-09-17 | 宁波大学 | Preparation method of protozoan cyst transmission electron microscope sample |
| CN113405869B (en) * | 2021-06-09 | 2024-02-27 | 宁波大学 | Preparation method of protozoan cyst transmission electron microscope sample |
| CN113418766A (en) * | 2021-06-24 | 2021-09-21 | 中国科学院华南植物园 | Double-fixation embedding sample preparation method after whole immunization |
| CN113418766B (en) * | 2021-06-24 | 2023-10-03 | 中国科学院华南植物园 | Double-fixation embedding sample preparation method after integral immunization |
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Application publication date: 20140507 |