CN109298174A - A kind of multiple-color immunofluorescence labeling method and imaging method - Google Patents
A kind of multiple-color immunofluorescence labeling method and imaging method Download PDFInfo
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Abstract
The invention discloses a kind of multiple-color immunofluorescence labeling method and imaging methods, are related to immunofluorescence technique field.The multiple-color immunofluorescence labeling method includes multiple fluorescent marker steps and the crosslinking fixing step that sample is incubated for cross linking fixative carried out after each fluorescent marker step；Cross linking fixative contains: acetone, paraformaldehyde and PBS buffer solution.It can be for a variety of different fluorescent dyes on a variety of antigenic marks in sample using the labeling method, sample after label can be used for confocal microscopic image, show a variety of different colors, and various colors it is clear, it is bright, do not alter color, this method is that researcher observes property, positioning and content of a variety of antigens etc. simultaneously and provides the foundation.
Description
Technical field
The present invention relates to immunofluorescence technique field, in particular to a kind of multiple-color immunofluorescence labeling method and at
Image space method.
Background technique
Immunofluorescence dyeing technology is the principle according to antigen-antibody reaction, first that known antigen or antibody label is upper glimmering
Fluorescent marker is made in light element, then use this fluorescence antibody (or antigen) as molecular probe check cell or tissue in it is corresponding
Antigen (or antibody).Contain fluorescein on the antigen antibody complex formed in cell or tissue, is seen using fluorescence microscope
Examine sample, the irradiation of fluorescein stimulated luminescence and issue bright fluorescence (yellow green or salmon pink), it can be seen where fluorescence
Cell or tissue, so that it is determined that the property of antigen or antibody, positioning, and using quantitative technique measure content.
But the dyeing type of existing immunofluorescence technique is limited, and it is more for a sample dyeing type when, hold
Easily there is the case where altering color.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of multiple-color immunofluorescence labeling methods, can be in sample using the labeling method
A variety of antigenic marks on a variety of different fluorescent dyes, the sample after label can be used for confocal microscopic image, shows more
Kind different colors, at most up to 20 kinds of colors, and various colors it is clear, it is bright, do not alter color.
Another object of the present invention is to provide a kind of multiple-color immunofluorescence imaging methods, use the imaging method can be with needle
The sample for marking a variety of different fluorescent dyes is imaged, shows a variety of different colors, at most reachable 20 kinds of colors,
And various colors it is clear, it is bright, do not alter color.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of multiple-color immunofluorescence labeling methods comprising: multiple fluorescent marker steps with
And the crosslinking fixing step carried out after each fluorescent marker step；
Fluorescent marker step includes: to be incubated for the sample after primary antibody is incubated for secondary antibody, wherein secondary antibody is marked with fluorescence dye
Material；
Fluorescent dye used in each fluorescent marker step is different, and primary antibody is combined anti-in each fluorescent marker step
It is former different；
Crosslinking fixing step includes: to be incubated for sample with cross linking fixative；
Wherein, cross linking fixative contains: acetone, paraformaldehyde and PBS buffer solution.
Multiple-color immunofluorescence labeling method provided by the invention, after each fluorescent marker, using containing acetone, paraformaldehyde
It is fixed with PBS buffer solution cross linking fixative, can be incited somebody to action by being repeated continuously fluorescent marker step and being crosslinked fixing step
A variety of fluorescent dyes are steadily tagged on the not synantigen in sample, can be by the glimmering of these labels by confocal microscopic image
Photoinitiator dye develops the color one by one, and the quantity of colour developing and the fluorescent dye classification of label are consistent, and the various colors shown is clear, bright
It is bright, do not alter color.This method observes property, positioning and content of a variety of antigens etc. simultaneously for researcher and provides the foundation.
Situations such as antigen classification for the observational study that the quantity of fluorescent marker step can be studied according to actual needs, determines.
For example, it is desired to study 10 kinds of antigens, then the quantity of fluorescent marker step can be 10, each fluorescent marker step is not using
The different antigen of same fluorochrome label, each fluorescent marker step all need once be crosslinked fixing step after the completion, hand over
The fluorescent marker step of next antigen is carried out after connection is fixed again.
Further, in some embodiments of the present invention, by volume percentage, cross linking fixative contains: 45%-
57% acetone, 0.3%-0.7% paraformaldehyde and 42.5%-54.5%PBS buffer.
Preferably, in some embodiments of the present invention, by volume percentage, cross linking fixative contains: 50% the third
Ketone, 0.5% paraformaldehyde and 49.5%PBS buffer.
Such as the preparation method of 100ml cross linking fixative is: taking 50ml acetone, 0.5ml paraformaldehyde and 49.5ml PBS
Buffer, mixing, obtains cross linking fixative.
The 1L of the PBS buffer solution is formulated: potassium dihydrogen phosphate (KH2PO4) 0.24g, disodium hydrogen phosphate (Na2HPO4) 1.44g,
Sodium chloride (NaCl) 8.0g, potassium chloride (KCl) 0.2g, adds water to 1000mL, pH7.4 or so.
Further, in some embodiments of the present invention, crosslinking fixing step includes: to be incubated for sample with cross linking fixative
Then this 4-6min cleans sample with PBS buffer solution, the sample after cleaning is for next fluorescent marker step or redyes core step
Suddenly.
After the completion of the last one fluorescent marker step, after crosslinked fixing step processing, then carry out redying core step.
It includes: to be incubated for sample with DAPI that this, which redyes core step, after being cleaned with PBS buffer solution, adds anti-fluorescent quenching mounting liquid
Carry out mounting.Sample after mounting can be used to imaging.
Further, in some embodiments of the present invention, fluorescent marker step includes:
Primary antibody is incubated for: being incubated for sample with primary antibody, then is cleaned with PBS buffer solution；
Secondary antibody is incubated for: after cleaning, being incubated for sample with secondary antibody；Then it is cleaned with PBS buffer solution, the sample after cleaning is for handing over
Join fixing step.
It should be understood that
Primary antibody is the first antibody of combining target antigen, and the target antigen being directed in different fluorescent marker steps is not
Together, therefore, the primary antibody that different fluorescent marker steps uses is also different；
Secondary antibody is the antibody of anti-primary antibody, and secondary antibody is marked with fluorescent dye, by and primary antibody combination, and then fluorescence is contaminated
On the target antigen for expecting label.Certainly, for the difference of displaying target antigen.In multiple fluorescent marker steps, any two
The classification of fluorescent dye used in fluorescent marker step is different, and has different emission spectrum as far as possible therebetween, with
Convenient for observation color with the not synantigen of separator.
Certainly, it should be noted that in some embodiments, in fluorescent marker step can directly be marked with fluorescence dye
The primary antibody of material is incubated for sample, omits the step of being incubated for again using secondary antibody.
Further, in some embodiments of the present invention, it in primary antibody incubation step, while being incubated using a variety of primary antibodies
Educate sample；Wherein, animal of a variety of primary antibodies from different genera, the different antigen of a variety of anti-bindings.
, in order to save time, can be in first order fluorescence markers step when needing to mark a variety of antigens, while using more
Kind primary antibody is incubated for sample.It is respectively incorporated to a variety of primary antibodies on corresponding target antigen.Compared in first order fluorescence markers step
A kind of method for only marking target antigen, while it being incubated for sample using a variety of primary antibodies, plurality of target antigen can be marked simultaneously, to the greatest extent
Possibly shorten the label time, reduces workload.It should be noted that these a variety of primary antibodies need to from the animal of different genera,
To avoid cross reaction.If it is a variety of primary antibodies from same kind, it is easy to appear cross reaction.
Correspondingly, after being incubated for sample using a variety of primary antibodies, in secondary antibody incubation step, it is also desirable to use the two of the same category
Resist to be incubated for again, be incubated for sample with a variety of secondary antibodies of a variety of primary antibodies are resisted respectively, a variety of secondary antibodies are correspondingly with a variety of one
It is anti-respectively in connection with and then will be on different fluorochrome label to different target antigens.Certainly, what a variety of secondary antibodies were marked is glimmering
Photoinitiator dye is also different each other.
Further, in some embodiments of the present invention, the animal in above-mentioned primary antibody source include but is not limited to mouse,
These animals such as rat, rabbit, goat, people, camel, horse, monkey, chicken or pig.As long as the animal that can produce antibody can make
For the source for the primary antibody that the present invention uses.
Further, in some embodiments of the present invention, fluorescent dye is selected from AF647, AF633, AF700, PE-
Cy7、AF532、AF568、BODIPY FL、Qdot625、AF514、PerCP-Cy5.5、Cy5.5、TRITC、Cy5、CFL680、
Cy3, Dylight405, AF488, Qdot655, Qdot585 and AF430.
Certainly, it should be noted that when using a variety of fluorescent dyes, the classifications of a variety of fluorescent dyes can with actual conditions into
Row combination.Certainly, fluorescent dye used in the present invention is not limited to above-mentioned fluorescent dye.
Further, in some embodiments of the present invention, sample is histotomy, frozen section or cell.
Further, in some embodiments of the present invention, multiple-color immunofluorescence labeling method further include: at first
The pre-treatment step carried out before fluorescent marker step；
Pre-treatment step includes: to clean sample with PBS buffer solution with paraformaldehyde fixed sample, then with closing rupture of membranes liquid
It is incubated for sample；
Wherein, closing rupture of membranes liquid is the lowlenthal serum containing Triton-100, and the condition of sample is incubated for closing rupture of membranes liquid
Are as follows: 35-38 DEG C of temperature, time 1.2-2h.
On the other hand, the present invention also provides a kind of multiple-color immunofluorescence imaging methods comprising: it will be through polychrome as above
Treated that sample is imaged with Laser Scanning Confocal Microscope for immunofluorescence label method.
The imaging method Laser Scanning Confocal Microscope to through multiple-color immunofluorescence labeling method as above treated sample into
The color of a variety of different fluorescent dyes can be distinguished, a variety of different colors be shown, at most up to 20 kinds by row imaging
Color, and various colors it is clear, it is bright, do not alter color.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is confocal microscopic image result of the embodiment of the present invention to the frozen section of C57/B6 mouse heart.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment marks the multiple-color immunofluorescence of the present embodiment using the frozen section of C57/B6 mouse heart as sample
Method is illustrated, specific as follows:
1. pretreatment
The frozen section of 1.1C57/B6 mouse heart fixes 15min with 4% paraformaldehyde, then cleans three with PBS buffer solution
It is secondary, each 5min.
After 1.2 cleanings, frozen section is with closing rupture of membranes liquid in 37 DEG C of incubation 1.5h.
Wherein, closing rupture of membranes liquid is lowlenthal serum, contains 0.3% Triton-100.
2. the 1st fluorescent marker
2.1 primary antibodies are incubated for: (being purchased from the Troponin T antibody of the combinable Troponin T albumen in mouse source
Abcam, specification 200 μ g, article No. ab8295) α-SMA of combinable α-SMA albumen in (press 1:10 dilution proportion) and rabbit source resists
Body (being purchased from Abcam, specification 10ul, article No. ab32575) (pressing 1:20 dilution proportion) and frozen section, are incubated in 4 DEG C
Overnight (> 10h).
2.2 cleaning frozen section twice with PBS buffer solution, each 5min after primary antibody is incubated for.
2.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse of the anti-Troponin T antibody marked with fluorescent dye AF647
It IgG (H+L) (be purchased from the green skies, specification 0.1ml, article No. A0473) (dilution ratio 1:10) and is marked with fluorescent dye AF633
Anti alpha-SMA antibody secondary antibody goat anti-rabbit igg (H+L) (be purchased from Invitrogen, specification 500ul, article No. A-21070) it is (dilute
Release ratio 1:10) and frozen section, 1h is incubated in 37 DEG C.
After 2.4 secondary antibodies are incubated for, frozen section is cleaned twice with PBS buffer solution, each 5min.
3. crosslinking is fixed
Frozen section after 2.4 step process is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS
Buffer solution for cleaning frozen section three times, each 5min.
The cross linking fixative contains: 50% acetone, 0.5% paraformaldehyde and 49.5%PBS buffer.
Described previously herein and related PBS buffer solution 1L hereinafter preparation method is as follows:
Take potassium dihydrogen phosphate (KH2PO4) 0.24g, disodium hydrogen phosphate (Na2HPO4) 1.44g, sodium chloride (NaCl) 8.0g, chlorine
Change potassium (KCl) 0.2g, adds water to 1000mL, sufficiently dissolve to obtain the final product, pH 7.4 or so.
The preparation method of cross linking fixative 100ml used in the present embodiment is as follows: taking 50ml acetone, 0.5ml poly first
Aldehyde and 49.5ml PBS buffer solution mix to get cross linking fixative used in the present embodiment.
3. the 2nd fluorescent marker
3.1 primary antibodies are incubated for: with the DDR2 antibody of combinable the DDR2 albumen in mouse source (purchased from Santa, specification 10ul,
Article No. sc-81707) (press 1:20 dilution proportion) and rabbit source combinable WT1 albumen WT1 antibody (purchased from Abcam, specification
10ul, article No. ab89901) (pressing 1:20 dilution proportion) and frozen section, it is incubated in 4 DEG C overnight (> 10h).
After 3.2 primary antibodies are incubated for, frozen section is cleaned twice with PBS buffer solution, each 5min.
3.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse IgG (H+ of the anti-DDR2 antibody marked with fluorescent dye AF700
L it) (be purchased from Invitrogen, specification 500ul, article No. A-21036) (dilution ratio 1:20) and is marked with fluorescent dye PE-Cy7
The anti-WT1 antibody of note secondary antibody goat anti-rabbit igg (be purchased from Santa, specification 10ul, article No. sc-516721) (dilution ratio 1:
20) and frozen section, 1h is incubated in 37 DEG C.
After 3.4 secondary antibodies are incubated for, frozen section is cleaned twice with PBS buffer solution, each 5min.
4. crosslinking is fixed
Frozen section after 3.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
5. the 3rd fluorescent marker
5.1 primary antibodies are incubated for: (being purchased from Abcam, rule with the Vimentin antibody of the combinable Vimentin albumen in mouse source
Lattice 100ul, article No. ab8978) the FSP1 antibody of combinable FSP1 albumen in (by 1:50 dilution proportion) and rabbit source (is purchased from
Abcam, specification 100ul, article No. ab197896) (press 1:50 dilution proportion) and frozen section, be incubated in 4 DEG C overnight (>
10h)。
After 5.2 primary antibodies are incubated for, frozen section is cleaned three times with PBS buffer solution, each 5min.
5.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse of the anti-Vimentin antibody marked with fluorescent dye AF532
IgG (H+L) (be purchased from Invitrogen, specification 500ul, article No. A-11002) (dilution ratio 1:50) and have fluorescent dye
The secondary antibody goat anti-rabbit igg (H+L) of the anti-FSP1 antibody of AF568 label (is purchased from Invitrogen, specification 500ul, article No. A-
11011) (dilution ratio 1:50) and frozen section are incubated with 1h in 37 DEG C.
After 5.4 secondary antibodies are incubated for, frozen section is cleaned three times with PBS buffer solution, each 5min.
6. crosslinking is fixed
Frozen section after 5.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
7. the 4th fluorescent marker
7.1 primary antibodies are incubated for: with the Thy1 antibody of combinable the Thy1 albumen in mouse source (purchased from Santa, specification 10ul,
Article No. sc-53116) (press 1:100 dilution proportion) and rabbit source combinable NG2 albumen NG2 antibody (purchased from Abcam, specification
100ul, article No. ab129051) (pressing 1:100 dilution proportion) and frozen section, it is incubated in 4 DEG C overnight (> 10h).
After 7.2 primary antibodies are incubated for, frozen section is cleaned three times with PBS buffer solution, each 5min.
7.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse of the anti-Thy1 antibody marked with fluorescent dye BODIPY FL
IgG (H+L) (be purchased from Invitrogen, specification 1mg article No. B-2752) (dilution ratio 1:100) and have fluorescent dye
Qdot625 label anti-NG2 antibody 2 goat anti-rabbit igg (H+L) of secondary antibody F (ab') (be purchased from Invitrogen, specification 100ul,
Article No. A-10194) (dilution ratio 1:100) and frozen section, 1h is incubated in 37 DEG C.
After 7.4 secondary antibodies are incubated for, frozen section is cleaned three times with PBS buffer solution, each 5min.
8. crosslinking is fixed
Frozen section after 7.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
9. the 5th fluorescent marker
9.1 primary antibodies are incubated for: (being purchased from Santa, specification with the PDGFR- β antibody of the combinable PDGFR- β albumen in mouse source
10ul, article No. sc-374573) the Tie2 antibody of combinable Tie2 albumen in (by 1:100 dilution proportion) and rabbit source (is purchased from
Abcam, specification 100ul, article No. ab221961) (pressing 1:100 dilution proportion) and frozen section, it is incubated in 4 DEG C overnight
(>10h)。
After 9.2 primary antibodies are incubated for, frozen section 4 times are cleaned with PBS buffer solution, each 5min.
9.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse IgG of the anti-PDGFR- β antibody marked with fluorescent dye AF514
(H+L) (be purchased from Invitrogen, specification 500ul, article No. A-31555) (dilution ratio 1:100) and fluorescent dye is had
The secondary antibody goat anti-rabbit igg (being purchased from Santa, specification 10ul, article No. sc-45101) of the anti-Tie2 antibody of PerCP-Cy5.5 label
(dilution ratio 1:100) and frozen section is incubated with 1h in 37 DEG C.
After 9.4 secondary antibodies are incubated for, frozen section 4 times are cleaned with PBS buffer solution, each 5min.
10. crosslinking is fixed
Frozen section after 9.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
11. the 6th fluorescent marker
11.1 primary antibodies are incubated for: (being purchased from the periostin antibody of the combinable periostin albumen in mouse source
Santa, specification 10ul, article No. sc-398631) (press 1:100 dilution proportion) and rat source combinable sca-1 albumen
Sca-1 antibody (being purchased from Ebioscience, specification 100ul, article No. 14-5981-81) (pressing 1:100 dilution proportion) and frost
Slice is incubated with overnight (> 10h) in 4 DEG C.
After 11.2 primary antibodies are incubated for, frozen section 2 times are cleaned with PBS buffer solution, each 5min.
11.3 secondary antibodies are incubated for: with the secondary antibody sheep anti-Mouse of the anti-periostin antibody marked with fluorescent dye Cy5.5
IgG (be purchased from Bo Aosen, specification 100ul, article No. bs-0296G-Cy5.5) (dilution ratio 1:100) and have fluorescent dye
The secondary antibody goat anti-rat IgG (being purchased from Zhong Shan Golden Bridge, specification 100ul, article No. ZF-0318) of the anti-sca-1 antibody of TRITC label
(dilution ratio 1:100) and frozen section is incubated with 1h in 37 DEG C.
After 11.4 secondary antibodies are incubated for, frozen section 2 times are cleaned with PBS buffer solution, each 5min.
12. crosslinking is fixed
Frozen section after 11.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
13. the 7th fluorescent marker
13.1 primary antibodies are incubated for: with the CD45 antibody of combinable the CD45 albumen in mouse source (purchased from Santa, specification 10ul,
Article No. sc-1178) the CD31 antibody of combinable CD31 albumen in (press 1:100 dilution proportion) and rabbit source (purchased from Abcam, advises
Lattice 100ul, article No. ab24590) (pressing 1:100 dilution proportion) and frozen section, it is incubated in 4 DEG C overnight (> 10h).
After 13.2 primary antibodies are incubated for, frozen section 3 times are cleaned with PBS buffer solution, each 5min.
13.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse IgG (purchase of the anti-CD45 antibody marked with fluorescent dye Cy5
From Bo Aosen, specification 100ul, article No. bs-0296G-Cy5) (dilution ratio 1:100) and with fluorescent dye CFL680 label
The secondary antibody goat anti-rabbit igg (being purchased from Santa, specification 10ul, article No. sc-516252) (dilution ratio 1:100) of 1 antibody of AntiCD3 McAb
And frozen section, 1h is incubated in 37 DEG C.
After 13.4 secondary antibodies are incubated for, frozen section 3 times are cleaned with PBS buffer solution, each 5min.
14. crosslinking is fixed
Frozen section after 13.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
15. the 8th fluorescent marker
15.1 primary antibodies are incubated for: (being purchased from Santa, specification with the c-kit antibody of the combinable c-kit albumen in mouse source
10ul, article No. sc-365504) (pressing 1:50 dilution proportion) and frozen section, it is incubated in 4 DEG C overnight (> 10h).
After 15.2 primary antibodies are incubated for, frozen section 4 times are cleaned with PBS buffer solution, each 5min.
15.3 secondary antibodies are incubated for: with the secondary antibody goat anti-mouse IgG (H+ of the anti-c-kit antibody marked with fluorescent dye Cy3
L) (dilution ratio 1:100) and frozen section (purchased from the green skies, specification 100ul, article No. A0521), are incubated in 37 DEG C
1h。
After 15.4 secondary antibodies are incubated for, frozen section 4 times are cleaned with PBS buffer solution, each 5min.
16. crosslinking is fixed
Frozen section after 15.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
17. the 9th fluorescent marker
17.1 primary antibodies are incubated for: (being purchased from Santa, specification with the PDGFR Alpha antibodies of the combinable PDGFR α albumen in mouse source
10ul, article No. sc-398206) (pressing 1:50 dilution proportion) and frozen section, it is incubated in 4 DEG C overnight (> 10h).
After 17.2 primary antibodies are incubated for, frozen section 4 times are cleaned with PBS buffer solution, each 5min.
17.3 secondary antibodies are incubated for: being resisted with the secondary antibody goat of the anti-PDGFR Alpha antibodies marked with fluorescent dye Dylight405 small
Mouse IgG (H+L) (be purchased from the green skies, specification 100ul, article No. A0609) (dilution ratio 1:100) and with fluorescent dye
WGA antibody (primary antibody is purchased from Sigma, specification 2mg, article No. L4895-2MG) (dilution of the combinable WGA albumen of AF488 label
Ratio 1:100) and frozen section, 1h is incubated in 37 DEG C.
After 17.4 secondary antibodies are incubated for, frozen section 4 times are cleaned with PBS buffer solution, each 5min.
18. crosslinking is fixed
Frozen section after 17.4 steps is placed in crosslinking fixating reagent, in incubation at room temperature 5min, then PBS is buffered
Liquid cleans frozen section three times, each 5min.
19. redying core
19.1 will through step 18 treated frozen section DAPI (being purchased from the green skies, specification 50ml, article No. C1006) in
10min is incubated under the conditions of 37 DEG C.
After 19.2 are incubated for, frozen section 5 times are cleaned with PBS buffer solution, each 5min.
After 19.3 cleanings, anti-fluorescent quenching mounting liquid (purchased from doctor's moral, specification 10ml, article No. AR1109) is added and is sealed
Piece, it is spare.
In above-mentioned correlation step, when primary antibody dilutes, (green cloud is purchased from PBST buffer or primary antibody diluted
It, specification 100ml, article No. P0103)；When secondary antibody dilutes, (the green skies, specification are purchased from PBST or secondary antibody diluent dilution
100ml, article No. P0108)；Wherein, the Triton-X 100 that the PBST of 1L is the addition 3ml in the PBS buffer solution of 1L is made into.
Embodiment 2
A kind of multiple-color immunofluorescence imaging method is present embodiments provided, using sample made from embodiment 1 as observation
Object is imaged with Laser Scanning Confocal Microscope (Leica SP5 confocal microscopy), and the specific operation method is as follows:
It is copolymerized burnt program:
With 405 laser, according to the light (taking pictures to 415-445nm range) of Dylight405；
With 405 laser, carries out spectral scan and (scan the wave-length coverage of 415-481nm, with 3nm for a scan unit, often
Increase 1nm and carry out single pass (all the same after the mode of spectral scan)) (split Dylight405 and DAPI) (give up
Dylight405 saves the figure of DAPI)；
With 405 laser, according to the light (taking pictures to 600-660nm range) of Qdot625；
With 488 laser, spectral scan (scanning 500-548nm) (splitting AF488, PE-Cy7 and BODIPY FL) is carried out；
With 514 laser, spectral scan (scanning 524-599nm) (splitting AF514, AF532, Cy3, TRITC) is carried out；
With 543 laser, according to the light (taking pictures to 569-631nm range) of AF568；
With 633 laser, carries out spectral scan (scanning 643-685nm) (splitting AF633, AF647, Cy5) and (give up Cy5, protect
Deposit the figure of AF633 and AF647)；
With 633 laser, according to the light (taking pictures to 675-685nm range) of Cy5；
With 633 laser, carries out spectral scan (scanning 685-739nm) (splitting CFL680, Cy5.5, AF700) and (give up
Cy5.5 saves the figure of CFL680 and AF700)；
With 633 laser, according to the light (taking pictures to 703-710nm range) of Cy5.5.
Using commonly take pictures and spectral scan by the way of, be avoided that Spectrographic scanning carry out spectrum fractionation when there is mistake
The case where, because the spectrum of part fluorescein is variant from theoretical spectrum atlas, splits some problems occurred when calculating.Its
His fluorescein combination is taken pictures, and need to formulate the burnt camera program of corresponding copolymerization in conjunction with actual spectrum situation.
As a result as shown in Figure 1, as can be seen from Figure 1:
(first figure (PDGFR α) RGB (83,0,255), second figure (DAPI) RGB (0,0,255), third figure
(NG2) (0,255,255) RGB, the 4th figure (WGA) RGB (0,255,0), the 5th figure (WT1) RGB (255 255 0), the
Six figure (Thy1) RGB (168,255,0), the 7th figure (PDGFR β) RGB (225,119,49), the 8th figure (c-kit) RGB
(255,78,0), the 9th figure (Vimentin) RGB (255,0,0), the tenth figure (sca-1) RGB (255,0,255), the tenth
One figure (FSP1) RGB (0,0,0), the 12nd figure (α-SMA) RGB (42,86,162), the 13rd figure (Troponin T)
RGB (120,185,85), the 14th figure (CD45) RGB (203,228,75), the 15th figure (Tie2) RGB (205,62,
51), the 16th figure (CD31) RGB (111,255,134), the 17th figure (DDR2) RGB (242,166,57), the 18th
Scheme (periotin) RGB (86,45,0), the 19th figure (Merge) is the figure of the synthesis of preceding 18 figures).Text below picture
Word is the antigen title of label.
As seen from Figure 1, by the sample of a variety of different fluorochrome labels, a variety of different face can be shown
Color, every kind of antigen protein mark different dyestuffs, show different colors, and up to 18 kinds of the present embodiment, antigen in 18
Albumen by different dye markers, and various colors it is clear, it is bright, do not alter color.In other examples up to 20 kinds, even
More, specific how many kinds of can be marked according to demand.
To sum up, the whole operation process of multiple-color immunofluorescence labeling method provided in an embodiment of the present invention by primary antibody, secondary antibody,
Repeatedly operating process is to solve the primary antibody in different genera source and the intersection of secondary antibody the sequence of cross linking fixative in batches
The problem of reaction.And in different fluorescent marker step operations, antibody is washed by certain number and detergent type, is subtracted
The influence combined to antibody is washed less.And in the way of taking pictures with the combining of spectral scan, it can solve some pure spectrums
Split undesirable situation.
In short, can be for a variety of different fluorescent dyes on a variety of antigenic marks in sample, mark using the labeling method
Sample after note can be used for confocal microscopic image, show a variety of different colors, at most reachable 20 kinds of colors, and various face
Color is clear, it is bright, do not alter color.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of multiple-color immunofluorescence labeling method, characterized in that it comprises: multiple fluorescent marker steps and each glimmering
The crosslinking fixing step carried out after signal step；
The fluorescent marker step includes: to be incubated for the sample after primary antibody is incubated for secondary antibody, wherein secondary antibody is marked with fluorescence dye
Material；
Fluorescent dye used in each fluorescent marker step is different, and primary antibody is combined anti-in each fluorescent marker step
It is former different；
The crosslinking fixing step includes: to be incubated for the sample with cross linking fixative；
Wherein, the cross linking fixative contains: acetone, paraformaldehyde and PBS buffer solution.
2. multiple-color immunofluorescence labeling method according to claim 1, which is characterized in that volume percentage is pressed, it is described
Cross linking fixative contains: 45%-57% acetone, 0.3%-0.7% paraformaldehyde and 42.5%-54.5%PBS buffer.
3. multiple-color immunofluorescence labeling method according to claim 2, which is characterized in that the crosslinking fixing step packet
It includes: being incubated for the sample 4-6min with the cross linking fixative, then clean the sample with PBS buffer solution, the institute after cleaning
Sample is stated for next fluorescent marker step or redyes core step.
4. multiple-color immunofluorescence labeling method according to claim 1-3, which is characterized in that the fluorescent marker
Step includes:
Primary antibody is incubated for: being incubated for the sample with primary antibody, then is cleaned with PBS buffer solution；
Secondary antibody is incubated for: after cleaning, being incubated for the sample with secondary antibody；Then it is cleaned with PBS buffer solution, the sample after cleaning is used
In crosslinking fixing step.
5. multiple-color immunofluorescence labeling method according to claim 4, which is characterized in that
The sample is incubated in primary antibody incubation step, while using a variety of primary antibodies；Wherein, a variety of primary antibodies are from different genera
Animal, the different antigen of a variety of anti-bindings.
6. multiple-color immunofluorescence labeling method according to claim 5, which is characterized in that the animal be mouse, rat,
Rabbit, goat, people, camel, horse, monkey, chicken or pig.
7. multiple-color immunofluorescence labeling method according to claim 1-3, which is characterized in that fluorescent dye is selected from
AF647、AF633、AF700、PE-Cy7、AF532、AF568、BODIPY FL、Qdot625、AF514、PerCP-Cy5.5、
Cy5.5, TRITC, Cy5, CFL680, Cy3, Dylight405, AF488, Qdot655, Qdot585 and AF430.
8. multiple-color immunofluorescence labeling method according to claim 1-3, which is characterized in that the sample is group
Knit slice, frozen section or cell.
9. according to the described in any item multiple-color immunofluorescence labeling methods of claim 8, which is characterized in that
The multiple-color immunofluorescence labeling method further include: the pre-treatment step carried out before first fluorescent marker step；
The pre-treatment step includes: to fix the sample with paraformaldehyde, cleans the sample with PBS buffer solution, then with envelope
It closes rupture of membranes liquid and is incubated for the sample；
Wherein, the closing rupture of membranes liquid is the lowlenthal serum containing Triton-100, is incubated for the sample with closing rupture of membranes liquid
Condition are as follows: 35-38 DEG C of temperature, time 1.2-2h.
10. a kind of multiple-color immunofluorescence imaging method, characterized in that it comprises: will be described in any item through claim 1-9
Treated that sample is imaged with Laser Scanning Confocal Microscope for multiple-color immunofluorescence labeling method.
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