CN103364570B - Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses - Google Patents
Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses Download PDFInfo
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- CN103364570B CN103364570B CN201310332662.4A CN201310332662A CN103364570B CN 103364570 B CN103364570 B CN 103364570B CN 201310332662 A CN201310332662 A CN 201310332662A CN 103364570 B CN103364570 B CN 103364570B
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Abstract
The invention discloses a method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses. The method comprises the following steps of carrying out fixing of an oosperm with a membrane, stripping a blastoderm of the fixed oosperm from a whole embryo by a dissecting needle, punching the stripped blastoderm, reducing or eliminating the influence produced by uncombined aldehyde groups on an experimental result by sodium borohydride, carrying out sample sealing and fluorescent double-labeling dyeing, and carrying out sample observation by a fluorescence microscope or a laser scanning confocal microscope. The method has low sample requirements, has simple processes, is convenient for observation, produces a clear result, has a high three-dimensional feel, can be used for dynamic change research on flounder oosperm microtubules and cell nucleuses from blastoderm formation respectively to 2-cell, 4-cell and 8-cell development stages, is suitable for other larger telolecithal eggs of marine flounder oosperms, and has a wide application range.
Description
Technical field
The present invention relates to immunocytochemical technique and fluorescence microscopy, be specifically related to the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton and nuclear fluorescence double-tagging.
Background technology
Immunocytochemistry is according to immunology principle, utilizes antibody antigen to be carried out to the technology of single-minded position-finding with specific antigen combination.If by antibody in conjunction with upper label, then with tissue in antigen react, can under light microscopic or Electronic Speculum, demonstrate this antigen and be present in the position in tissue.Therefore, this technology is by the general designation of some research methods of the micro-or sub-microhistology of selectivity reaction bonded between antigen, antibody and complement in immunology, is the combination of immunology principle and light microscopic or electron microscopy.Fluorescence microscopy is can present the fluorescence of certain color, then location and the distribution situation in cell in this cell component of fluorescence microscopy Microscopic observation after utilizing specific fluorescent dye special component in cell to be combined.Fluorescence double-tagging microexamination technology refers to utilizes the fluorescence labeling technology technical method of the interior two kinds of biomacromolecules of labeled cell simultaneously.The applied research of fluorescence double-tagging microexamination technology aspect egg cell skeleton and nucleus, more aspect mammal, mainly concentrates on mouse, pig, rabbit etc.Aspect fruit bat and Xenopus laevis (Xenopus), also there iing relevant report.In the application aspect sea life egg cell skeleton and nucleus, mainly concentrate on the aspects such as sea urchin, sea cucumber and prawn.The applied research of fluorescence double-tagging microexamination technology aspect fish egg cell skeleton and nucleus is less, current rarely seen blue or green Jiang (Abraham etc., 1995) and zebra fish (Suresh etc., 1996; KarenW.Lee etc., 2004; Robin E. Lindeman etc., 2012) fresh-water fishes such as, so far there are no the report of the applied research aspect fish egg cell skeleton and nucleus.
Because the immunofluorescence label technology of zygote microtubule protein molecular is utilized Ag-Ab specific reaction and is carried out, so the application of this labelling technique aspect egg cell, all need first egg membrane to be removed, so that the corresponding large molecular antibody of tubulin enters, egg cell is inner is combined with corresponding antigens, then carries out mark and microexamination.Above-mentioned reported utilize fluorescence double-tagging microexamination technical research fertilized egg cell's skeleton and nuclear correlative study to be first egg membrane is removed, then be fixed, fluorescence labeling and microexamination.Seawater left-eyed flounder after fertilization, protoplasm forms more flat and thin discoid blastodisc in embryonated egg animal pole, and protoplasm is finer and close; And fertilization membrane is thin and tough and tensile, the ovum week gap between itself and vitellinae membrana is narrow visible hardly.Be difficult to the fertilization membrane of the seawater left-eyed flounder zygote living to remove, then the embryonated egg of striping is fixed.Therefore, in the equal yellow embryonated egg of routine (mammal) the early embryonic development process of having reported, the fluorescence double-tagging microexamination technical method of tubulin and DNA is restricted in the application aspect seawater left-eyed flounder zygote, need to set up the fluorescence double-tagging microscopy observation method of tubulin and DNA in a kind of seawater left-eyed flounder zygote early embryonic development process.
Summary of the invention
For above-mentioned fluorescence double-tagging microexamination technology at the application restric-tion aspect seawater left-eyed flounder zygote microtubule skeleton and nucleus, the invention provides the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton and nuclear fluorescence double-tagging, the method is first with immobile liquid, sample to be fixed, under common anatomical lens, pick out again the embryonated egg of developmental condition compared with normal, with tip tweezers, blastodisc is peeled off out from whole embryonated egg, and then punch, sealing and fluorescence double-tagging, finally with fluorescent microscope or laser scanning co-focusing microscope, observe and take pictures.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
A microscopy observation method for seawater left-eyed flounder zygote microtubule skeleton and nuclear fluorescence double-tagging, it comprises the following steps:
(1) seawater left-eyed flounder zygote is placed in to formaldehyde-glutaraldehyde immobile liquid room temperature and fosters 3-6 hour, directly corresponding with the displacement of phosphoric acid tween buffer solution
formaldehyde-glutaraldehyde immobile liquid of volume, and in 4 ℃ of preservations, stand-by;
(2) under anatomical lens, pick out the embryonated egg of developmental condition compared with normal, and with dissecting needle, blastodisc is peeled off out from whole embryonated egg;
(3) with Triton-100 to the sample processing of punching, then use NaBH
4solution is fostered sample three times, is no more than half an hour at every turn;
(4) adopt improved indirect immunofluorescence labeling method step to carry out mark to sample, the basic step of indirect immunofluorescence mark is: seal → foster primary antibodie → clean → foster two anti-→ clean, detailed process is as follows:
Sealing: at room temperature with confining liquid, sample is sealed and processes 1-2 hour, described confining liquid is PBS+0.1% Tween-20+1%DMSO+10% lowlenthal serum;
Foster primary antibodie: first with antibody diluent, monoclonal mouse-anti alpha-tubulin is diluted and makes primary antibodie suspending liquid, then by the sample after sealing in primary antibodie suspending liquid 4 ℃ foster and spend the night, described antibody diluent is to contain the Tris hydrochloride buffer that percentage is the dimethyl sulfoxide (DMSO) of 10% lowlenthal serum and 5%;
Clean: sample is cleaned 3-4 time with cleaning fluid, and each 10 minutes, described cleaning fluid was PBS+0.1%Tween-20;
Foster two anti-: with described antibody diluent, the goat anti-mouse IgG of Alexa Fluor 555 marks is diluted, make two and resist and foster liquid, add DNA fluorescent dye DAPI simultaneously, then under lucifuge condition, sample room temperature is fostered to 0.5-1 hour,
With cleaning fluid, clean 5-6 time more each 10 minutes;
(5) make self-contained of embryo, with fluorescent microscope or laser scanning co-focusing microscope, observe and take pictures.
Further, described step
in fixing embryonated egg do not carry out striping processing.
Further, described step
formaldehyde-glutaraldehyde immobile liquid formula of middle use is: 80 mM K-PIPES, 5 mM EGTA, 100 mM MgCl
2, 400 mM NaCl, 3.7% formaldehyde, 0.25% glutaraldehyde and 0.5%Triton X-100.
Further, described step
the composition of middle phosphoric acid tween buffer solution is: 128 mM NaCl, 2 mM KCl, 8 mM NaH
2pO
4, 2 mM KH
2pO
4, 0.1% Tween-20, pH 7.2.
Further, described step
middle is 25-30 ℃ with Triton-100 to the sample temperature of processing of punching, and concentration is 4-5%, and the processing time is 10-12 hour.
Further, described step
middle NaBH
4solution is the 2.5mg/L solution of 50% ethanol preparation.
Further, in described step (4), with antibody diluent, monoclonal mouse-anti alpha-tubulin is diluted, dilution ratio is 1:500-1000.
Further, described step
middle is 1:250-500 by the ratio that antibody diluent dilutes the goat anti-mouse IgG of Alexa Fluor 555 marks; In the DNA fluorescent dye adding, to account for the ratio of cumulative volume be 1:10000-100000 to DAPI.
Further, described step
during self-contained of middle making embryo, add anti-fluorescence quenching, make fluorescence signal stronger, the holding time is more lasting.
Compare with existing left-eyed flounder zygote fluorescence labeling technology, advantage of the present invention and good effect are as follows:
1. in invention, fixed solution is improved:
Former fixed solution: embryonated egg room temperature in immobile liquid was fostered after 3-6 hour, with phosphoric acid tween buffer solution (PBST) (PBST:128 mM NaCl, 2 mM KCl, 8 mM NaH
2pO
4, 2 mM KH
2pO
4, 0.1% Tween-20, pH 7.2) clean twice, finally 4 ℃ of preservations in PBST, stand-by.
Improve fixed solution: embryonated egg room temperature in immobile liquid was fostered after 3-6 hour, directly used phosphoric acid tween buffer solution (PBST) (PBST:128 mM NaCl, 2 mM KCl, 8 mM NaH
2pO
4, 2 mM KH
2pO
4, 0.1% Tween-20, pH 7.2) replace corresponding
the immobile liquid of volume, and in 4 ℃ of preservations, stand-by.
Fixed solution after improvement is conducive to the long-term preservation of fixing rear embryonated egg.
2. in invention, adopt dissecting needle that the blastodisc of the embryonated egg after fixing is peeled off out from whole ovum, make immunofluorescence technique be able to applying aspect the cytoskeleton research of seawater left-eyed flounder zygote on the one hand; Avoided on the other hand the impact on experimental result when observing of yolk and oily ball, blastodisc has been peeled off out from whole ovum simultaneously and also can make the macromolecular immunohistochemical study of other biological of such embryonated egg be carried out.
3. working concentration and temperature and the sodium borohydride (NaBH of Triton-100 of processing in invention punch sample
4) processing scheme of solution carried out corresponding improvement.
Former scheme: under normal temperature with percentage 1-2% Triton-100 to the sample processing of punching, then containing 80-200mM NaBH
4in phosphate buffer solution (PBS), under room temperature, foster 6-16 hour,
Improvement project: at 25-30 ℃ of temperature with the Triton-100 of 4-5% to the sample processing of punching.2.5mg/L sodium borohydride (the NaBH configuring with 50% ethanol again
4) solution fosters sample three times, is no more than half an hour at every turn.
Processing scheme after improvement can more effectively be eliminated the impact of unconjugated aldehyde radical on test findings, makes fluorescence signal stronger, and background is lower, and the microtubule skeleton of embryonated egg and nucleus structure more clearly display.
4. in invention, the two anti-processes of fostering in indirect immunofluorescence labeling method step have been carried out to corresponding improvement: in two of the goat anti-mouse IgG that contains Alexa Fluor 555 marks (H+L), anti-add DNA fluorescent dye DAPI in fostering liquid simultaneously.
Disposal route after improvement can be carried out mark to the tubulin of embryonated egg and DNA molecular simultaneously, is convenient to dynamic change and the mutual relationship thereof of research and inquirement tubulin and DNA molecular, for the correlative study of its molecular cytobiology provides technical support.
5. after mark, the observation disposal route of sample is improved: the sample by mark and after cleaning adds anti-fluorescence quenching, and uses nail polish mounting, observes after making self-contained again.
Through improving, make self-contained of embryo and both can directly at the fluorescence microscopy Microscopic observation of just putting, take pictures, also can carry out scanning with inverted laser scanning co-focusing microscope.Utilize the continuous optical section of the not damaged of laser scanning co-focusing microscope and from continuous section, construct 3-D view function, first carrying out continuous Multi Slice Mode and observe, then construct 3 D stereo conformation, making test findings picture clear, stereoscopic sensation is strong.Can also utilize the multicolor fluorescence image collecting function of confocal microscope, adopt the fluorescent dual labelling technique of tubulin and DNA, common location and the interaction of tubulin and DNA in research left-eyed flounder zygote early embryonic development process.In addition, when film-making, add anti-fluorescence quenching, can effectively prevent fluorescent quenching, both handled easilies, also can make fluorescence signal stronger, keep more lasting.
6. having wide range of applications of the method, can be used for being fused to from both sexes the immunocytochemical study of the developmental stage embryonated eggs such as 2-cell, 4-cell, 8-cell, is applicable to other larger anisolecithal eggs of similar seawater left-eyed flounder zygote simultaneously.
Accompanying drawing explanation
Fig. 1 is the fluorescence double-tagging microexamination picture of turbot 1-interphase cell zygote microtubule albumen of the present invention and nucleus DNA.
Fig. 2 is the fluorescence double-tagging microexamination picture of turbot 2-cell zygote microtubule in mid-term albumen of the present invention and nucleus DNA.
Fig. 3 is the fluorescence double-tagging microexamination picture of turbot 4-cell zygote microtubule in mid-term albumen of the present invention and nucleus DNA.
Fig. 4 is the fluorescence double-tagging microexamination picture of lefteye flounder 1-cell zygote microtubule albumen in early stage of the present invention and nucleus DNA.
Embodiment
Below in conjunction with drawings and Examples in detail technical scheme of the present invention is described in detail.
Principle of the present invention is: the immunofluorescence technique in fluorescence Double Labelling Technique is to utilize Ag-Ab specific reaction to study specified protein or nucleic acid at the effective ways at tissue, cells time and position.Its ultimate principle is to utilize the antibody of specified protein, it is first antibody, make it with histotomy or self-contained in this kind of protein bound, then the antibody that adds fluorescein-labeled anti-first antibody, be second antibody, with suitable incident light, observe under the microscope, because the combination of Ag-Ab is highly narrow spectrum, the visible fluorescence just at the position with this kind of protein only, expression rule that thus can Study on Protein.Fluorescence labeling technology is to utilize specific fluorescent dyestuff to be combined with DNA, observes under the microscope with suitable incident light, because this combination is highly narrow spectrum, only, at this position ability visible fluorescence, can study nuclear expression rule thus.
The fluorescence double-tagging microscopy observation method of embodiment 1 turbot 1-interphase cell zygote microtubule albumen and nucleus DNA
1, adopt formaldehyde-glutaraldehyde immobile liquid of Pipes preparation to be fixed sample, the turbot fertilized eggs of striping room temperature in immobile liquid was not fostered after 3 hours, directly with phosphoric acid tween buffer solution (PBST) (PBST:128 mM NaCl, 2 mM KCl, 8 mM NaH
2pO
4, 2 mM KH
2pO
4, 0.1% Tween-20, pH 7.2) replace corresponding
the immobile liquid of volume, and in 4 ℃ of preservations, stand-by.
2, under anatomical lens, pick out the embryonated egg of developmental condition compared with normal, and with dissecting needle, blastodisc is peeled off out from whole embryonated egg.
3, at 25 ℃ of temperature, the Triton-100 with 4% punches and processes 12 hours sample.2.5mg/L sodium borohydride (the NaBH configuring with 50% ethanol again
4) solution fosters sample three times, is no more than half an hour at every turn, reduce or eliminate the impact of unconjugated aldehyde radical on experimental result.
4, adopt improved indirect immunofluorescence labeling method step to carry out mark to sample.The basic step of indirect immunofluorescence mark is: seal → foster primary antibodie → clean → foster two anti-→ clean.Detailed process is as follows:
Sealing: at room temperature use confining liquid (confining liquid: PBS+0.1% Tween-20+1%DMSO+10% lowlenthal serum) sealing is processed 2 hours to sample.
Foster primary antibodie: first use antibody diluent (antibody diluent is to contain the Tris hydrochloride buffer that percentage is the dimethyl sulfoxide (DMSO) of 10% lowlenthal serum and 5%) that monoclonal mouse-anti alpha-tubulin (α-Tubulin) is diluted to (ratio is 1:1000) and make primary antibodie suspending liquid, then by the sample after sealing in primary antibodie suspending liquid 4 ℃ foster and spend the night.
Clean: cleaning fluid for sample (PBST:PBS+0.1%Tween-20) is cleaned 3 times to each 10 minutes.
Foster two to resist: with antibody diluent, the goat anti-mouse IgG of Alexa Fluor 555 marks (H+L) to be diluted to (dilution ratio 1:500), make the two anti-liquid of fostering, add DNA fluorescent dye DAPI(dilution ratio 1:100000 simultaneously), then under lucifuge condition, sample room temperature is fostered to 0.5-1 hour.
With cleaning fluid, clean 6 times more each 10 minutes.
5, make self-contained of embryo and observe and take pictures: the blastodisc after cleaning is drawn with suction pipe, be added drop-wise on clean microslide, after being blotted, solution adds appropriate anti-fluorescence quenching, covered, use nail polish mounting, dry and with fluorescent microscope or laser scanning co-focusing microscope, observe and take pictures afterwards.
The fluorescence double-tagging microexamination process of embodiment 2, turbot 2-cell zygote microtubule in mid-term albumen and nucleus DNA
Operation steps is substantially with embodiment 1, and difference from Example 1 is:
Turbot fertilized eggs time of fostering in formaldehyde-glutaraldehyde immobile liquid is 6 hours;
Developmental stage is 2-cell mid-term, and the temperature that Triton-100 punching is processed is 30 ℃, and concentration is 5%, and the processing time is 12 hours;
Monoclonal mouse-anti alpha-tubulin (α-Tubulin) dilution ratio is 1:750;
Goat anti-mouse IgG (H+L) dilution ratio of Alexa Fluor 555 marks is 1:500;
DAPI dilution ratio is 1:10000.
The fluorescence double-tagging microexamination process of embodiment 3, turbot 4-cell zygote microtubule in mid-term albumen and nucleus DNA
Operation steps is substantially with embodiment 1, and difference from Example 1 is:
Turbot fertilized eggs is fostered 5 hours time in formaldehyde-glutaraldehyde immobile liquid;
Developmental stage is 4-cell mid-term, and the temperature that Triton-100 punching is processed is 28 ℃, and concentration is 4%, and the processing time is 10 hours;
Monoclonal mouse-anti alpha-tubulin (α-Tubulin) dilution ratio is 1:500;
Goat anti-mouse IgG (H+L) dilution ratio of Alexa Fluor 555 marks is 1:250;
DAPI dilution ratio is 1:10000.
The fluorescence double-tagging microexamination process of embodiment 4, lefteye flounder 1-cell zygote microtubule albumen in early stage and nucleus DNA.
Operation steps is substantially with embodiment 1, and difference from Example 1 is:
Lefteye flounder embryonated egg is fostered 4 hours time in formaldehyde-glutaraldehyde immobile liquid;
Developmental stage is 1-cell early stage, and the temperature that Triton-100 punching is processed is 28 ℃, and concentration is 5%, and the processing time is 12 hours;
Monoclonal mouse-anti alpha-tubulin (α-Tubulin) dilution ratio is 1:500;
Goat anti-mouse IgG (H+L) dilution ratio of Alexa Fluor 555 marks is 1:500;
DAPI dilution ratio is 1:100000.
From Fig. 1-Fig. 4, can find out, Fig. 1 a of the present invention, Fig. 2 a, Fig. 3 a, Fig. 4 a can be clearly seen that the mark result (as shown by arrows) of tubulin (α-Tubulin); From Fig. 1 b, Fig. 2 b, Fig. 3 b, Fig. 4 b, can be clear that the mark result (as shown by arrows) of nucleus (DNA), a and b stack from Fig. 1 c, Fig. 2 c, Fig. 3 c, Fig. 4 c(figure) can see the double-tagging result (as shown by arrows) of tubulin (α-Tubulin) and nucleus (DNA).
Claims (6)
1. a microscopy observation method for seawater left-eyed flounder zygote microtubule skeleton and nuclear fluorescence double-tagging, is characterized in that it comprises the following steps:
(1) seawater left-eyed flounder zygote is placed in to formaldehyde-glutaraldehyde immobile liquid room temperature and fosters 3-6 hour, directly corresponding with the displacement of phosphoric acid tween buffer solution
formaldehyde-glutaraldehyde immobile liquid of volume, and in 4 ℃ of preservations, stand-by;
(2) under anatomical lens, pick out the embryonated egg of developmental condition compared with normal, and with dissecting needle, blastodisc is peeled off out from whole embryonated egg;
(3) with Triton-100 to the sample processing of punching, temperature is 25-30 ℃, concentration is 4-5%, the processing time is 10-12 hour, then uses NaBH
4solution is fostered sample three times, is no more than half an hour at every turn; Described NaBH
4solution is the 2.5mg/L solution of 50% ethanol preparation;
(4) adopt improved indirect immunofluorescence labeling method step to carry out mark to sample, the basic step of indirect immunofluorescence mark is: seal → foster primary antibodie → clean → foster two anti-→ clean, detailed process is as follows:
Sealing: at room temperature with confining liquid, sample is sealed and processes 1-2 hour, described confining liquid is PBS+0.1% Tween-20+1%DMSO+10% lowlenthal serum;
Foster primary antibodie: first with antibody diluent, monoclonal mouse-anti alpha-tubulin is diluted and makes primary antibodie suspending liquid, then by the sample after sealing in primary antibodie suspending liquid 4 ℃ foster and spend the night, described antibody diluent is to contain the Tris hydrochloride buffer that percentage is the dimethyl sulfoxide (DMSO) of 10% lowlenthal serum and 5%;
Clean: sample is cleaned 3-4 time with cleaning fluid, and each 10 minutes, described cleaning fluid was PBS+0.1%Tween-20;
Foster two to resist: with described antibody diluent, the goat anti-mouse IgG of Alexa Fluor 555 marks to be diluted, make the two anti-liquid of fostering, add DNA fluorescent dye DAPI simultaneously, then under lucifuge condition, sample room temperature is fostered to 0.5-1 hour, with cleaning fluid, clean 5-6 time more each 10 minutes;
(5) make self-contained of embryo, with fluorescent microscope or laser scanning co-focusing microscope, observe and take pictures; While making self-contained of embryo, add anti-fluorescence quenching, covered, uses nail polish mounting, makes fluorescence signal stronger, and the holding time is more lasting.
2. the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton according to claim 1 and nuclear fluorescence double-tagging, is characterized in that: in described step (1), fixing embryonated egg is not carried out striping processing.
3. the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton according to claim 1 and nuclear fluorescence double-tagging, it is characterized in that: the formaldehyde-glutaraldehyde immobile liquid formula using in described step (1) is: 80 mM K-PIPES, 5 mM EGTA, 100 mM MgCl
2, 400 mM NaCl, 3.7% formaldehyde, 0.25% glutaraldehyde and 0.5%Triton X-100.
4. the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton according to claim 1 and nuclear fluorescence double-tagging, it is characterized in that: in described step (1), the composition of phosphoric acid tween buffer solution is: 128 mM NaCl, 2 mM KCl, 8 mM NaH
2pO
4, 2 mM KH
2pO
4, 0.1% Tween-20, pH 7.2.
5. the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton according to claim 1 and nuclear fluorescence double-tagging, it is characterized in that: in described step (4), with antibody diluent, monoclonal mouse-anti alpha-tubulin is diluted, dilution ratio is 1:500-1000.
6. the microscopy observation method of a kind of seawater left-eyed flounder zygote microtubule skeleton according to claim 1 and nuclear fluorescence double-tagging, is characterized in that: in described step (4), by the ratio that antibody diluent dilutes the goat anti-mouse IgG of Alexa Fluor 555 marks, be 1:250-500; In the DNA fluorescent dye adding, to account for the ratio of cumulative volume be 1:10000-100000 to DAPI.
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