CN102072959A - Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors - Google Patents
Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors Download PDFInfo
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Abstract
The invention discloses a method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors. The method comprises the following steps: 1. immobilizing tissue sections on a plus microscope slide processed by polylysine; 2. dewaxing the tissue sections in dimethylbenzene; 3. carrying out antigen retrieval; 4. washing the sections; 5. carrying out confining; 6. adding primary antibodies; 7. washing the sections; 8. carrying out confining in the same way as the step 5; 9. using the fragment antigen-binding F(ab')2-QDs525 of goat anti-mouse immunoglobulin G labelled by quantum dot QDs525, the fragment antigen-binding F(ab')2-QDs585 of goat anti-rabbit immunoglobulin G labelled by quantum dot QDs585 and the fragment antigen-binding F(ab')2-QDs655 of rabbit anti-goat immunoglobulin G labelled by quantum dot QDs655 as secondary antibodies and dropwise adding the mixture after removing the confining liquid; 10. washing the sections; and 11. sealing the sections: preparing buffered glycerol with glycerol and 10ml of tris buffered saline (TBS) and storing the sections after sealing the sections with the buffered glycerol. The method is used for efficiently, accurately, rapidly and simultaneously detecting various components in tumor tissue microenvironment.
Description
Technical field
The invention belongs to histogenic immunity fluorescent technique field, more specifically relate to a kind of detection mesenchyma stroma of tumors IV Collagen Type VI, macrophage and tumor neovasculature quantum dot three fluorescence labeling method, be applicable to accurate, detection by quantitative that people's tumor tissues microenvironment composition changes.
Background technology
The generation of tumour, development not only relate to tumour cell itself, but also with tumour cell microenvironment on every side substantial connection are arranged.Malignant tumour (tumour) microenvironment is by tumour cell and fibroblast, epithelial cell, intrinsic and specific immunity cell, tumor vessel, mesenchymal cell and expression product thereof, metabolic product on every side.Composition is numerous in the tumor microenvironment, technology effectively for want of, current research to tumor microenvironment can't show the multiple change in the tumor microenvironment simultaneously still based on single component, takes place as the reinventing of mesenchyma stroma of tumors, macrophages infiltration and tumor vessel.
Numerous and the interactional characteristic prompting research tumor microenvironment of tumor microenvironment composition must have good labelling technique can study multiple composition simultaneously.The paraffin section immunohistochemical staining is the mark specific molecular to a certain extent, and is significant in the tumour pathological study.But this method dyeing background height, poor specificity, and traditional enzyme-substrate Color Appearance System depends on the deposition of chromogen are very approximate organizationally, are difficult to observe simultaneously the coexpression location of the two kinds of antigens in a same position of section.This limitation makes it can't satisfy the needs of tumour multicomponent mark.Though immunofluorescence label antigen also is the common method of research tumour, and more have superiority than enzyme-substrate display system, but fluorescein isothiocynate (fluorescein isothiocyanate, FITC), rhodamine common organic fluorescence group susceptibilitys such as (rhodamine) is low, spectral width takes place and overlapping, anti-photobleaching ability, and be difficult to distinguish with the high autofluorescence of tissue.In addition, in the two mark of traditional immunofluorescence decoration method, organic dyestuff must excite under different wave length, utilizes image processing software to carry out image overlay then, could obtain the two mark of synthetic last fluorescence coloration result.Therefore, common organic fluorescent dye can not satisfy the needs of multiple staining.
Quantum dot is the subsphaeroidal nano particle of diameter 2 ~ 10nm, radius less than or approach the novel semi-conductor nanocrystal of exciton Bohr radius, compare with traditional organic fluorescent dye, fluorescin and rare earth chelate compound, quantum dot has many advantages, excitation light wave spectrum width, continuous, ultraviolet light can excite the quantum dot of any size, and emission light wave spectrum is narrow and symmetrical; Good light stability.These characteristics make quantum dot have special advantages in the bio-imaging field, have broad application prospects in tumor research.
Summary of the invention
The object of the present invention is to provide the method for matter IV Collagen Type VI, macrophage and new vessels between a kind of while marked tumor, the quantum dot multiple staining method that provides among the present invention can be used for detecting efficiently, accurately, fast, simultaneously of the multiple composition of tumor tissues microenvironment, and this method can be promoted and be used for simple, quick, sensitive, efficient, the economical and practical general tumor microenvironment molecular pathology detection technique of a kind of operation steps.
The method of matter IV Collagen Type VI, macrophage and new vessels the steps include: between a kind of while marked tumor
A, the thick section of the paraffin-embedded tumor tissues 4 μ m of formalin fixed are fixed on the anticreep microslide that poly-D-lysine is handled.
The preparation of B, histotomy, histotomy is put into three times (different vessel) of dimethylbenzene dewaxing, each 4-6 minute, after the dewaxing histotomy put into successively absolute ethyl alcohol 4-6 minute, 95%(volume ratio, below identical) alcohol 1-3 minute, 95% alcohol 1-3 minute and 80% alcohol 1-3 minute, flowing water flushing 3 ~ 5 minutes.
C, antigen retrieval, use trisodium citrate 29.41g, distilled water 1000ml preparation trisodium citrate damping fluid, citric acid 10.5g, distilled water 500 ml prepare citrate buffer solution, getting trisodium citrate damping fluid 20.25ml, citrate buffer solution 4.75ml and 225ml distilled water respectively is mixed with citric acid antigen retrieval liquid (0.01mol, pH 6.0,250ml).Histotomy is placed the antigen retrieval box microwave reparation that has added 250ml antigen retrieval liquid, promptly low fire (microwave power: 136W) repair and go to moderate heat after 4-6 minute (microwave power: 440W) repaired 9-11 minute, room temperature (identical below 20 ~ 25 ℃) is placed and treated that slide naturally cools to 36-38 ℃ then.
Section is taken out in D, tap water flushing back, washing slice, with trihydroxy aminomethane (tris (hydroxymethyl) aminomethane, Tris) 6.05g and sodium chloride 38.0g and distilled water 5000ml, TBS (the tris buffered saline of preparation 0.01M/L, pH7.2-7.4, TBS), wash each 3 minutes 3 times as cleansing solution;
E, sealing, the sealing liquid level contains the TBS of 2%wt/vol bovine serum albumin(BSA), and sealing condition is: wet box is hatched, and 37 ℃, 30min;
F, add one anti-, after removing above-mentioned confining liquid, drip an anti-potpourri of mouse-anti human macrophage monoclonal antibody, rabbit anti-human IV type collagen polyclonal antibody, the anti-people CD105 of goat polyclonal antibody, hatch 3 ~ 4h or 4 ℃ of refrigerator overnight (being no less than 12h) in 37 ℃ of wet boxes; Described one anti-immunoglobulin G (Ig G) Fab for correspondence.
G, washing slice, cleaning fluid are (TBS(TBST that contains 0.5%/vol/vol tween #20), wash 3 times, each 3min;
H, sealing are with step 4;
Used two anti-are quantum dot (F (ab ') in I, this experiment
2-) the anti-rat immune globulin G(IgG of QDs525 labelled goat) and Fab (Fragment antigen-binding, Fab section) quantum dot (F (ab ')
2-) QDs525, quantum dot (F (ab ')
2-) the anti-rabbit immunoglobulin G(IgG of QDs585 labelled goat) and Fab (Fragment antigen-binding, Fab section) quantum dot (F (ab ')
2-) QDs585, quantum dot (F (ab ')
2-) Fab (Fragment antigen-binding, Fab the section) (F (ab ') of the anti-goat immunoglobulin G of rabbit (IgG) of QDs655 mark
2-QDs655), (F (ab ')
2-QDs525), (F (ab ')
2-QDs585) and (F (ab ')
2-QDs655) all available from American I nvitrogen company.After removing above-mentioned confining liquid, drip F (ab ')
2-QDs525, F (ab ')
2-QDs585 and F (ab ')
2-QDs655 potpourri, dilution are the TBS that contains the 2%wt/vol bovine serum albumin(BSA), and wet box is hatched, 37 ℃, and 2 ~ 3h.Described two anti-are quantum dot (F (ab ')
2-) the anti-rat immune globulin G(IgG of QDs525 labelled goat) and Fab (Fragment antigen-binding, Fab section) quantum dot (F (ab ')
2-) QDs525, quantum dot (F (ab ')
2-) the anti-rabbit immunoglobulin G(IgG of QDs585 labelled goat) and Fab (Fragment antigen-binding, Fab section) quantum dot (F (ab ')
2-) QDs585, quantum dot (F (ab ')
2-) Fab (Fragment antigen-binding, Fab the section) (F (ab ') of the anti-goat immunoglobulin G of rabbit (IgG) of QDs655 mark
2-QDs655).
J, washing slice, cleaning fluid is identical with above-mentioned steps 7 used cleaning fluids, and TBS washs 2 times by the formulation of above-mentioned steps 4, each 2min, more once with the TBS washing, 3min.
K, mounting are made into the 90%(volume ratio with glycerine 90ml and TBS10ml) buffering glycerine, 4 ℃ of preservations after the buffering glycerine mounting.(330 ~ 385nm) excite the tumor neogenetic blood vessels that promptly can be observed the macrophage that shows green fluorescence, the IV Collagen Type VI that shows orange-yellow fluorescence and demonstration red fluorescence with ultraviolet light under fluorescent microscope.
Can carry out quantitative analysis in order to detect this technology, do following experiment:
According to above-mentioned step, pass through fluorescent microscope, (wavelength 330 ~ 385nm) excites down, has obtained IV Collagen Type VI, macrophage and tumor neovasculature tricolor marker imaging (Fig. 5 A), and institutional framework and antigen distribution position be clear, be easy to distribute at ultraviolet light.Adopt CRi Nuance multi-optical spectrum imaging system (Cabridge Research ﹠amp; Instrumentation, Inc, Woburn, MA USA) gathers spectral signal (Fig. 5 B), can obtain more clearly image three-colo(u)r (Fig. 5 C), also can be to different fluorescence signals quantitative (Fig. 5 D-E).Macrophage both can directly count, and 13 macrophages are arranged in Fig. 5.The IV Collagen Type VI can be observed its distribution trend, and quantitative according to fluorescence intensity, it is divided into 186 continuous its expressions of regional quantitative Analysis in Fig. 5.Tumor neogenetic blood vessels also can direct census, and 14 new vesselses are arranged in Fig. 5.
The quantum dot multiple staining method that provides among the present invention can be used for detecting efficiently, accurately, fast, simultaneously of the multiple composition of tumor tissues microenvironment, and this method can be promoted and be used for simple, quick, sensitive, efficient, the economical and practical general tumor microenvironment molecular pathology detection technique of a kind of operation steps.
The present invention compared with prior art has the following advantages and effect:
1. detection specificity is strong, and background is clear, is convenient to techtology analysis (Fig. 1).
2. method is easy, quick, is easy to standardization and a large amount of the detection, and repeatability better.Dewax to observations from section, experiment the entire process have about 7 ~ 8h, can dye to many histotomies simultaneously.
3. to observe the multiple composition in the tumor microenvironment, can only dye separately when adopting traditional immunohistochemical staining, can't realize the coexpression location (Fig. 4) in the same section.The present invention has solved an inferior difficult problem effectively, can observe multiple composition simultaneously, and the analysis-by-synthesis microenvironment changes.
The detection sensitivity height, be convenient to quantize.Quantum dot fluorescence brightness is strong, stable and lasting, can adopt spectral analysis quantized result (Fig. 5).
5. need not complex analyses steps such as the later stage picture synthesizes, can Direct observation after the mounting.
6. fluorescent brightness height, longer duration, the result preserves easily and observes repeatedly.
Description of drawings
Fig. 1 is that the monochromatic labelling method of a kind of quantum dot shows important component synoptic diagram in the tumor microenvironment.
A is for resisting the anti-rabbit igg Fab of quantum dot QDs585 labelled goat section (F (ab ') with specificity rabbit anti-human IV type collagen antibody as one among Fig. 1
2-QDs585) as two anti-mark results, matter reinvents between the demonstration tumor microenvironment.
B is for resisting the anti-goat IgG Fab section of the rabbit of quantum dot QDs655 mark (F (ab ') with the anti-people CD105 of specificity goat antibody as one among Fig. 1
2-QDs655) as two anti-mark results, show tumor neogenetic blood vessels.
C is for resisting the anti-mouse IgG of quantum dot QDs525 labelled goat Fab section (F (ab ') with the mouse-anti human macrophage as one among Fig. 1
2-QDs525) as two anti-mark results, show and soak into macrophage.
D is for resisting the anti-goat IgG Fab section of the rabbit of quantum dot QDs655 mark (F (ab ') with TBS as one among Fig. 1
2-QDs655) as two anti-mark results, prove labelling technique high specificity based on the quantum dot probe.
[enlargement factor: 200 * (A, B), 400 * (C, D); Scale: 50 μ m (A, B), 20 μ m (C, D). the quantum dot image is excited down by ultraviolet light (wavelength 330 ~ 385 nm), gathers image by Olympus DP72 imaging system]
Fig. 2 shows the result schematic diagram of three kinds of compositions in the tumor microenvironment simultaneously for a kind of quantum dot tricolor marker.
Fig. 3 shows the result schematic diagram of three kinds of compositions in the tumor microenvironment simultaneously for a kind of quantum dot tricolor marker.
The potpourri that adopts mouse-anti human macrophage monoclonal antibody, rabbit anti-human IV type collagen polyclonal antibody, the anti-people CD105 of goat polyclonal antibody in the experiment is one anti-, F (ab ')
2-QDs525, F (ab ')
2-QDs585 and F (ab ')
2The potpourri of-QDs655 is two anti-.Fig. 2 shows abundant new vessels is arranged between two cancer nests and soak into macrophage, and the important component IV Collagen Type VI of a matter then is degraded, and matter is reinvented between generation.Fig. 3 shows that along with the amplification of tumour cell, a matter barrier fades away, and visible macrophage is assembled at the cancer nests periphery in this process, and cancer nests periphery new vessels is abundant.
[enlargement factor: 400 * (Fig. 2, Fig. 3); Scale: 20 μ m (Fig. 2, Fig. 3).Excite down at ultraviolet light (wavelength 330 ~ 385 nm), gather image] by Olympus DP72 imaging system
Fig. 4 shows the result schematic diagram of three kinds of compositions in the tumor microenvironment respectively for a kind of traditional immunohistochemical staining.
These three compositions are dyeed simultaneously the variation of analysis-by-synthesis tumor microenvironment composition again after therefore can only dyeing respectively because of being implemented in the same section.
A is for anti-as one with specificity rabbit anti-human IV type collagen antibody among Fig. 4, biotinylated goat anti-rabbit igg is two anti-, streptomysin avidin-peroxidase is a reactant, diaminobenzidine (Diaminobenzidine, DAB) be the note result of chromogenic substrate, show matter composition between tumor microenvironment.
B is for anti-as one with the anti-people CD105 of specificity goat antibody among Fig. 4, the anti-goat IgG of biotinylated rabbit is two anti-, streptomysin avidin-peroxidase is a reactant, diaminobenzidine (Diaminobenzidine, DAB) be the note result of chromogenic substrate, show tumor neogenetic blood vessels.
C is for anti-as one with the mouse-anti human macrophage among Fig. 4, biotinylated mountain sheep anti-mouse igg is two anti-, and streptomysin avidin-peroxidase is a reactant, diaminobenzidine (Diaminobenzidine, DAB) be the note result of chromogenic substrate, show and soak into macrophage.
D is for anti-as one with TBS among Fig. 4, and biotinylated mountain sheep anti-mouse igg is two anti-, and streptomysin avidin-peroxidase is a reactant, and (Diaminobenzidine DAB) be the note result of chromogenic substrate to diaminobenzidine, organizes in contrast.
[enlargement factor: 400 * (A, B, C, D); Scale: 20 μ m (A, B, C, D).Excite down at ultraviolet light (wavelength 330 ~ 385 nm), gather image] by Olympus DP72 imaging system
Fig. 5 is a kind of quantitative spectrochemical analysis tricolor marker result's a synoptic diagram
A is the result schematic diagram that a kind of quantum dot tricolor marker shows three kinds of compositions in the tumor microenvironment simultaneously among Fig. 5.The potpourri that adopts mouse-anti human macrophage monoclonal antibody, rabbit anti-human IV type collagen polyclonal antibody, the anti-people CD105 of goat polyclonal antibody in the experiment is one anti-, F (ab ')
2-QDs525, F (ab ')
2-QDs585 and F (ab ')
2The potpourri of-QDs655 is two anti-.
The F (ab ') that is separated when B is for the employing quantitative spectrochemical analysis among Fig. 5
2-QDs525, F (ab ')
2-QDs585 and F (ab ')
2-QDs655 spectral signal.
C is for according to the synthesising picture behind the signal that B separated colouration again among Fig. 5.
D is to isolated F (ab ') among Fig. 5
2-QDs525 signal carries out the result schematic diagram of detection by quantitative.
E is to isolated F (ab ') among Fig. 5
2-QDs585 signal carries out the result schematic diagram of detection by quantitative.
F is to isolated F (ab ') among Fig. 5
2-QDs655 signal carries out the result schematic diagram of detection by quantitative.
[enlargement factor: 400 * (C D), excites down at ultraviolet light (wavelength 330 ~ 385 nm), gathers image by Olympus DP72 imaging system and CRi Nuance multi-optical spectrum imaging system for A, B.】
Embodiment
Embodiment 1:
The method of matter IV Collagen Type VI, macrophage and angiogenic the steps include: between a kind of while marked tumor
1. the thick section of the paraffin-embedded stomach organization 4 μ m of formalin fixed is fixed on the anticreep microslide that poly-D-lysine is handled.
2. the preparation of histotomy, histotomy is put into three times (different vessel) of dimethylbenzene dewaxing, each 5 minutes, after the dewaxing histotomy put into absolute ethyl alcohol successively 5 minutes, 95% alcohol 2 minutes, 95% alcohol 2 minutes and 80% alcohol 2 minutes, flowing water flushing 3 ~ 5 minutes.
3. antigen retrieval, with trisodium citrate 29.41g(Chemical Reagent Co., Ltd., Sinopharm Group), distilled water 1000ml preparation trisodium citrate damping fluid, citric acid 10.5g(Chemical Reagent Co., Ltd., Sinopharm Group), distilled water 500 ml prepare citrate buffer solution, getting trisodium citrate damping fluid 20.25ml, citrate buffer solution 4.75ml and 225ml distilled water respectively is mixed with citric acid antigen retrieval liquid (0.01mol, pH 6.0,250ml).Histotomy is placed antigen retrieval box (Bioisystech Co., Ltd of Beijing China fir Golden Bridge) microwave that has added 250ml antigen retrieval liquid, and (beautiful micro-wave oven-MK823ESJ-PA) repair (low fire reparation goes to moderate heat and repaired 10 minutes after 5 minutes), room temperature is placed and is treated the slide natural cooling then.
4. washing slice, be Chemical Reagent Co., Ltd., Sinopharm Group with trihydroxy aminomethane (Tris) 6.05g and sodium chloride 38.0g() and distilled water 5000, the TBS of preparation 0.01M/L, pH7.2-7.4 washs 3 times as cleansing solution, each 3min;
5. sealing, the sealing liquid level contains the TBS of 2%wt/vol bovine serum albumin(BSA) (Shanghai Ru Ji biotechnology Development Co., Ltd), and sealing condition is: wet box is hatched, 37 ℃ of (GNP-9080 type water isolation type constant incubators, the grand experimental facilities of last Nereid company limited), 30min;
6. traditional add one anti-, after removing above-mentioned confining liquid, drip mouse-anti human macrophage monoclonal antibody (MA1-38069, ABR, USA), rabbit anti-human IV type collagen polyclonal antibody (ab-6586, Abcam, England), the anti-people CD105 of goat polyclonal antibody (sc-13595, Santa Cruz, (dilution ratio of three kinds of antibody is respectively 1:200 to anti-potpourri USA), 1:50 and 1:50, dilution is TBS), wet box is hatched, 37 ℃ of (GNP-9080 type water isolation type constant incubator, the grand experimental facilities of last Nereid company limited) 3 ~ 4h or 4 ℃ of refrigerator overnight (being no less than 12h);
7. washing slice, cleaning fluid is TBST (TBS that contains 0.5%/vol/vol tween #20), washs 3 times, each 3min;
8. sealing is with step 4;
9. Cell immunohistochemical staining method dyeing background height, poor specificity are head it off, and this experiment is adopted and the corresponding immunoglobulin G of an anti-kind (Ig G) Fab (Fragment antibody binding, F (ab ')
2Section) the anti-and developer (see below and state) as two has reduced non-specific adsorption and the cross reaction of IgG FC (Fragment crystallizable, Fc section) in the tissue staining process.Used two anti-are the anti-rat immune globulin G(IgG of quantum dot QDs525 labelled goat in this experiment) Fab (Fragment antigen-binding, Fab section) (F (ab ')
2-QDs525), the anti-rabbit immunoglobulin G(IgG of quantum dot QDs585 labelled goat) Fab (Fragment antigen-binding, Fab section) (F (ab ')
2-QDs585), Fab (Fragment antigen-binding, Fab the section) (F (ab ') of the anti-goat immunoglobulin G of rabbit (IgG) of quantum dot QDs655 mark
2-QDs655), all available from Invitrogen company.After removing above-mentioned confining liquid, drip F (ab ')
2-QDs525, F (ab ')
2-QDs585 and F (ab ')
2-QDs655 potpourri (three kind of two anti-dilution ratio is 1:100, and dilution is the TBS that contains the 2%wt/vol bovine serum albumin(BSA)), wet box is hatched, 37 ℃ (GNP-9080 type water isolation type constant incubator, the grand experimental facilities of last Nereid company limited), 2 ~ 3h.
10. washing slice, TBST washing 2 times, each 2min, more once with the TBS washing, 3min.
11. mounting is made into 90% buffering glycerine with glycerine 90ml and TBS10ml, gets final product observations after the buffering glycerine mounting.Traditional enzyme-substrate Color Appearance System depends on the deposition of chromogen, and is very approximate organizationally, is difficult to observe simultaneously the coexpression location of the two kinds of antigens in a same position of section.And in the two mark of traditional immunofluorescence decoration method, organic dyestuff must excite under different wave length, utilizes image processing software to carry out image overlay then, could obtain the two mark of synthetic last fluorescence coloration result.But present technique adopts the colour developing of novel nano fluorescent material quantum dot, and (330 ~ 385nm) promptly can be observed macrophage, IV Collagen Type VI that shows orange-yellow fluorescence that shows green fluorescence and the tumor neogenetic blood vessels that shows red fluorescence after exciting through ultraviolet light under fluorescent microscope.Realized multiple antigen (IV Collagen Type VI, macrophage, CD105) is carried out multiple color (green F (ab ')
2-QDs525, orange-yellow F (ab ')
2-QDs585 and red F (ab ')
2-QDs655) mark need not image overlay and processing, has realized the What You See Is What You Get of experimental result, has simplified multi-color marking result's acquiring way, has reduced the bias that Flame Image Process causes.
Carry out the immunofluorescence multiple staining with quantum dot, under the prerequisite of susceptibility height, high specificity, show the variation characteristics of multiple composition in the tumor microenvironment simultaneously, and have paraffin section immunohistochemical staining histocyte clear in structure, Antigen Location characteristic of accurate.By quantum dot immune fluorescent three mark methods, shown in the tumor microenvironment simultaneously between matter reinvent the process that macrophages infiltration, tumor vessel take place.This method is applicable to paraffin-embedded any tumor tissues, the more important thing is, adopts this method can be easy, quick, sensitive and exactly tumor microenvironment is analyzed, and need not later steps such as picture is synthetic.
Claims (1)
1. the method for a while marked tumor IV Collagen Type VI, macrophage and new vessels the steps include:
A, the thick section of the paraffin-embedded tumor tissues 4 μ m of formalin fixed are fixed on the anticreep microslide that poly-D-lysine is handled;
The preparation of B, histotomy, histotomy is put into dimethylbenzene dewaxing three times, each 4-6 minute, after the dewaxing histotomy put into successively absolute ethyl alcohol 4-6 minute, 95% volume ratio alcohol 1-3 minute, 95% volume ratio alcohol 1-3 minute and 80% volume ratio alcohol 1-3 minute, flowing water flushing 3 ~ 5 minutes;
C, antigen retrieval, use trisodium citrate 29.41g, distilled water 1000ml preparation trisodium citrate damping fluid, citric acid 10.5g, distilled water 500 ml prepare citrate buffer solution, get trisodium citrate damping fluid 20.25ml, citrate buffer solution 4.75ml and 225ml distilled water respectively and be mixed with citric acid antigen retrieval liquid: 0.01mol, pH 6.0,250ml, histotomy is placed the antigen retrieval box microwave reparation that has added 250ml antigen retrieval liquid, low fire is repaired and is gone to moderate heat reparation 9-11 minute after 4-6 minute, and room temperature is placed and treated the slide natural cooling then;
Section is taken out in D, tap water flushing back, washing slice, with trihydroxy aminomethane 6.05g and sodium chloride 38.0g and distilled water 5000ml, the TBS of preparation 0.01M/L, pH7.2-7.4 is as cleansing solution, wash each 3 minutes 3 times;
E, sealing, the sealing liquid level contains the TBS of 2%wt/vol bovine serum albumin(BSA), and sealing condition is: wet box is hatched, and 37 ℃, 30min;
F, add one anti-, remove above-mentioned confining liquid after, drip an anti-potpourri of mouse-anti human macrophage monoclonal antibody, rabbit anti-human IV type collagen polyclonal antibody, the anti-people CD105 of goat polyclonal antibody, hatch 3 ~ 4h or 4 ℃ of refrigerator overnight in 37 ℃ of wet boxes;
G, washing slice, cleaning fluid are the TBS that contains 0.5%/vol/vol tween #20, wash 3 times, each 3min;
H, sealing, same step (D);
I, with the Fab F (ab ') of the anti-goat immunoglobulin G of rabbit of the Fab quantum dot QDs585 of two anti-Fab quantum dot QDs525, the anti-rabbit immunoglobulin G of quantum dot QDs585 labelled goat for the anti-rat immune globulin G of quantum dot QDs525 labelled goat, quantum dot QDs655 mark
2-QDs655, remove above-mentioned confining liquid after, drip quantum dot QDs525, quantum dot QDs585 and quantum dot QDs655 potpourri, dilution is the TBS that contains the 2%wt/vol bovine serum albumin(BSA), wet box is hatched, 37 ℃, 2 ~ 3h;
J, washing slice, cleaning fluid is identical with the used cleaning fluid of step (G), and TBS is the formulation of (D) set by step, washs 2 times, each 2min, more once with the TBS washing, 3min;
K, mounting, be made into 90% volume ratio buffering glycerine with glycerine 90ml and TBS10ml, 4 ℃ of preservations after the buffering glycerine mounting excite the tumor neogenetic blood vessels of observing the macrophage that shows green fluorescence, the IV Collagen Type VI that shows orange-yellow fluorescence and demonstration red fluorescence with ultraviolet light 330 ~ 385nm under fluorescent microscope.
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