CN103994911B - A kind of Double-immunofluorescent labeling method based on same Species origin first antibody - Google Patents
A kind of Double-immunofluorescent labeling method based on same Species origin first antibody Download PDFInfo
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- CN103994911B CN103994911B CN201410099873.2A CN201410099873A CN103994911B CN 103994911 B CN103994911 B CN 103994911B CN 201410099873 A CN201410099873 A CN 201410099873A CN 103994911 B CN103994911 B CN 103994911B
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Abstract
The invention discloses a kind of method that utilization carries out Double-immunofluorescent labeling with Species origin first antibody, carry out in accordance with the following steps: (1) gradient dilution anti-A protein antibodies, utilize indirect IF staining method, determine that anti-A protein antibodies detects the minimum working concentration of A albumen to be measured；(2) utilize this concentration, use SABC method (with fluorescein labelled streptavidin) that A albumen is developed the color；(3) apply anti-B protein antibodies, utilize indirect IF staining method, B albumen is developed the color；(4) fluorescence microscopy, take pictures, obtain the microscopy photo of A and B albumen to be measured and be overlapped, analyze two kinds of albumen expression.The present invention is simple and easy to do, easy and simple to handle, realizes higher research requirement with relatively low experimental cost, is widely applied scope for the research offer of clinical immunization fluorescence histochemistry.
Description
Technical field
The invention belongs to biomedicine and biological technology application, a kind of utilize two kinds of same Species origins
First antibody carries out the method for Double-immunofluorescent labeling.
Background technology
Biological with medical research, immunofluorescence dyeing is one of the most commonly used technological means, and its principle is
Utilizing the principle that antigen and antibody are specific binding, incorporation of markings fluorescein on antibody, by fluorescein stimulated luminescence
Irradiate and send bright fluorescence (yellow green or salmon pink) so that tissue, intracellular polypeptides or the location of proteantigen,
The information such as qualitative, quantitative visualizes under fluorescence microscope.
In research process, it is often necessary to detect two kinds of antigen/protein in same cell or tissue sample (such as simultaneously
A albumen and B albumen), with clear and definite they common location or coexpression situation, the most just need to apply Double-immunofluorescent labeling skill
Art, it can be divided into direct method and indirect method: (1) direct method is that (anti-A protein antibodies and anti-B albumen resist by two strain specific antibodies
Body) it is marked sending the fluorescein of different colours respectively, such as, anti-A protein antibodies fluorescein isothiocynate (FITC)
Mark sends yellow-green fluorescence, and anti-B protein antibodies TRITC (TRITC) mark sends orange red glimmering
Light, after being mixed two by proper proportion by fluorescence antibody, forms antigen antibody complex after hatching tissue or cell, shows at fluorescence
Select two kinds to excite filter disc to observe accordingly under micro mirror respectively, send the most anti-A protein antibodies binding site of yellow-green fluorescence, send out
Go out the position that the most anti-B protein antibodies of fluorescent red-orange combines, thus can demonstrate the position of two kinds of antigen/protein, simultaneously
Can be come target antigen/protein quantification by analysis of fluorescence intensity.The advantage of direct method is easy to be convenient, but sensitivity is relatively low,
Antibody requirement is big；(2) indirect method is first antibody (the A egg as anti-in mouse originated with the different genera of unmarked fluorescein
Bai Kangti and rabbit source property anti-B protein antibodies) hatch tissue or cell after, add the SA being marked with different fluoresceins
(such as anti-mouse IgG-FITC or anti-rabbit IgG-TRITC), thus two kinds of antigen/protein are positioned, quantitatively.Indirect method is because leading to
Cross SA so that the intensity of mark is amplified, so effect is sensitive, need antibody less, the most substantially instead of
Direct method, is the main method of immunofluorescence label and double labeling.
Developed recently goes out a kind of Double-immunofluorescent labeling method SABC method and indirect method combined.SABC i.e. strepto-
Avidin-Biotin-compound (Strept-avidin biotin complex), biotin and Avidin have natural height
Affinity.During experiment, the first antibody (A protein antibodies as anti-in mouse and rabbit source property anti-B protein antibodies) in different genera source
After being combined with A and B antigen/protein respectively, biotin labeled SA (such as anti-mouse IgG-biotin) is utilized to hatch tissue
Or cell, add Streptavidin (SA-Cy2) and anti-rabbit IgG-TRITC of fluorescein Cy2 mark afterwards, thus two kinds are resisted
Former/albumen develops the color.Owing to having 4 biotin-binding site, the amplification to fluorescence signal of the SABC method on 1 Avidin molecule
Effect is 5-10 times of simple indirect method, therefore has higher sensitivity.
Apply Double-immunofluorescent labeling technology time, it has to be noted that a bit: two species specificity first antibodies must come
Come from different genera, the most anti-A protein antibodies source mouse, then anti-B protein antibodies must derive from other kinds beyond mouse, as
Rabbit, goat or cavy etc., at this moment use the SA matched with first antibody kind just not have immunological cross-reaction,
Thus ensure the reliability of result.But in real work, the Species origin of antibody can be subject to many limitations, and is particularly grinding
Study carefully a kind of recruit or because search time and funds limit, it is impossible to select the kind of antibody, it is necessary to use two kinds to come with kind
During the first antibody in source, current Double-immunofluorescent labeling method exists for certain limitation.
Summary of the invention
The present invention is to solve that existing Double-immunofluorescent labeling technology must use two kinds of different genera sources first to resist
The problem of body, it is provided that a kind of new immunofluorescence staining technique, utilizes limited antibody Species origin and common fluorescent to show
Micro mirror realizes the double labeling in same sample of the first antibody with Species origin.
The present invention adopts the following technical scheme that realization:
A kind of Double-immunofluorescent labeling method based on same Species origin first antibody, it is with two kinds of same Species origins
Anti-A protein antibodies and anti-B protein antibodies the A albumen in sample to be detected and B albumen are carried out Double-immunofluorescent labeling.
Specifically, it is to utilize SABC immunofluorescence technique and IIF, at anti-A protein antibodies and anti-B albumen
When antibody is same Species origin, A albumen or B albumen to cell or tissue carry out double labeling detection.
A kind of Double-immunofluorescent labeling technicalization learns a skill, and utilizes two kinds of first antibodies with Species origin: anti-A albumen
Antibody and anti-B protein antibodies, comprise the following steps:
(1) anti-A protein antibodies minimum application concentration is determined:
A histotomy to be measured or cell climbing sheet are entered 0.01 mol/L PBS three times by (), each 5 minutes；
B () serum is closed；
C () the two times of corresponding first antibody of gradient dilution A albumen anti-A protein antibodies, join histotomy to be measured
Or cell climbing sheet, hatch 24-48 hour for 4 DEG C；
D () PBS three times, after each 5 minutes, adds the fluorescein mark second for first antibody Species origin and resists
Body, incubated at room 2 hours；
(e) PBS twice, 80% glycerol buffer mounting, fluorescence microscopy, find out and just can not separately show by A albumen
The first antibody minimum working concentration of look；
(2) SABC method detection A albumen:
A histotomy to be measured or cell climbing sheet are entered PBS three times by (), each 5 minutes；
B () serum is closed；
C () dilutes anti-A protein antibodies to minimum working concentration, join histotomy or cell climbing sheet, hatch 24-for 4 DEG C
48 hours；
D () PBS three times, after each 5 minutes, adds biotin labeled for anti-A protein antibodies Species origin
SA, incubated at room 3 hours；
E () PBS three times, after each 5 minutes, adds fluorescein-labeled Streptavidin, incubated at room 2 hours；
(f) PBS twice, fluorescence microscopy, observe coloration result；
(3) indirect method detection B albumen:
A histotomy that () PBS A albumen has developed the color or cell climbing sheet, after each 5 minutes, add anti-B albumen and resist
Body, hatches 24 hours for 4 DEG C；
B () PBS three times, after each 5 minutes, adds fluorescein-labeled SA, incubated at room 2 hours；
(c) PBS twice, 80% glycerol buffer mounting, fluorescence microscopy, observe coloration result；
(4) fluorescence picture overlay analysis:
A () fluorescence microscope is taken pictures, obtain the microscopy photo of testing protein A and B；
B () utilizes software to be overlapped photo, the coexpression of two kinds of albumen of qualitative and quantitative analysis and common positioning scenarios.
Double-immunofluorescent labeling technology of the present invention: utilize the principle that SABC method is sensitive compared with indirect method, at anti-A egg
Bai Kangti is in the case of minimum working concentration (can not develop the color for indirect method under this concentration), can develop the color A albumen；At B egg
When developing the color in vain, owing to utilizing indirect method, can only to B albumen but A albumen can not be developed the color, therefore avoid same kind derived antibodies
Between cross reaction.On the one hand this technology can be prevented effectively from the immunological cross-reaction of same Species origin first antibody, advances
Double-immunofluorescent labeling technology；On the other hand can solve antibody Species origin limited and because search time, funds are nervous nothing
Method selects a difficult problem for first antibody kind.
Accompanying drawing explanation
Fig. 1 a is the microscopy photo of A albumen (TH) after SABC method dyes, and Fig. 1 b is B albumen (P75) after indirect method dyeing
Microscopy photo, Fig. 1 c is the microscopy photo after A albumen (TH) superposes with B albumen (P75)；
Fig. 2 a is the microscopy photo of A albumen (Ki67) after SABC method dyes, and Fig. 2 b is B antigen (BrdU) after indirect method dyeing
Microscopy photo, Fig. 2 c is the microscopy photo after A albumen (Ki67) superposes with B antigen (BrdU).
Detailed description of the invention
Further illustrate the present invention by specific examples below, but be not limited to the scope of the present invention.
Embodiment l: newborn mice midbrain nucleus ceruleus plane frozen tissue section, uses immunofluorescence of the present invention dual
Labelling technique detection TH(A albumen) and P75(B albumen) coexpression situation, the first antibody of use is rabbit source property, including with
Lower step:
(1) rabbit anti-TH antibody minimum working concentration is determined:
A () newborn mice midbrain nucleus ceruleus plane frozen tissue section, enters 0.01 mol/L PBS three times after drying,
Each 5 minutes；
B () adds Normal Goat Serum confining liquid, hatch 60 minutes in 37 DEG C of wet boxes.Note keeping sample to moisten, necessarily
Histotomy to be avoided is dried, and otherwise can produce higher background；
C (), according to rabbit anti-TH antibody (Sigma company, article No. T-8700) specification recommended density, (contains with antibody diluent
1% BSA and 0.3% Triton-X100) two times of gradient dilution antibody, concrete extension rate is 1:500,1:1000,1:2000,
1:4000,1:8000, join histotomy to be measured, hatch 24-48 hour for 4 DEG C；
D () PBS three times, after each 5 minutes, adds anti-rabbit IgG-diluted by proper proportion with antibody diluent
TRITC, incubated at room 2 hours；
(e) PBS twice, 80% glycerol buffer mounting, fluorescence microscopy, find rabbit during 1:500 and 1:1000
The colour developing of anti-TH antibody is substantially；Remain to colour developing during 1:2000, but be obviously reduced；During 1:4000, antibody can not develop the color, and therefore 1:
The 4000 minimum working concentrations being rabbit anti-TH antibody；
(2) SABC method detection TH(A albumen)；
A () newborn mice midbrain nucleus ceruleus plane frozen tissue section, enters 0.01 mol/L PBS three times after drying,
Each 5 minutes；
B () adds Normal Goat Serum confining liquid, hatch 60 minutes in 37 DEG C of wet boxes；
C () is pressed 1:4000 with antibody diluent and is diluted rabbit anti-TH antibody, join histotomy 4 DEG C and hatch 24-48 hour；
D () PBS three times, after each 5 minutes, adds anti-rabbit IgG-diluted by proper proportion with antibody diluent
Biotin, incubated at room 3 hours；
E () PBS three times, after each 5 minutes, adds fluorescein-labeled Streptavidin (SA-Cy2), room temperature is kept away
Light hatches 2 hours；
(f) PBS twice, fluorescence microscopy, observe coloration result；
(3) indirect method detection P75(B albumen)；
A histotomy that () PBS TH has developed the color, each 5 minutes, adds and dilutes by proper proportion antibody diluent
Rabbit anti-P75 antibody (Abcam company, article No. ab38335), hatch 24 hours for 4 DEG C；
B () PBS three times, after each 5 minutes, anti-rabbit IgG-TRITC that addition dilutes with antibody diluent, room temperature is kept away
Light hatches 2 hours；
(c) PBS twice, 80% glycerol buffer mounting, fluorescence microscopy；
(4) fluorescence picture overlay analysis；
A () fluorescence microscope is taken pictures, obtain rabbit anti-TH antibody and the stained photographs of rabbit anti-P75 antibody；
B () utilizes software to be overlapped photo, result as shown in Figure 1: Fig. 1 a Green fluorescence represents purpose A albumen
(TH), Fig. 1 b red fluorescence represents purpose B albumen (P75), and Fig. 1 c is the superposition picture of the two, the cell coexpression shown in arrow
TH and P75, the cell shown in asterisk only expresses P75.
Embodiment 2: the NSC creep plate of cultivation, uses Double-immunofluorescent labeling technology for detection of the present invention
Ki67(A albumen) and BrdU(B antigen) coexpression situation, the first antibody of use is little mouse, comprises the following steps:
(1) little mouse-anti Ki67 antibody minimum working concentration is determined:
A NSC creep plate that () cultivates is after fixing 20 minutes in 4% paraformaldehyde, clear with 0.01 mol/L PBS
Wash three times, each 5 minutes；
B () adds Normal Goat Serum confining liquid, hatch 30 minutes in 37 DEG C of wet boxes；
C (), according to little mouse-anti Ki67 antibody (Abcam company, article No. ab6526) specification recommended density, dilutes with antibody
Two times of gradient dilution antibody of liquid, concrete extension rate is 1:200,1:400,1:800,1:1500,1:3000, join to be measured carefully
Born of the same parents' creep plate, hatches 24 hours for 4 DEG C；
D () PBS three times, after each 5 minutes, adds the anti-mouse IgG-by the dilution of proper proportion antibody diluent
TRITC, incubated at room 2 hours；
(e) PBS twice, 80% glycerol buffer mounting, fluorescence microscopy, find during 1:200 and 1:400 little
The colour developing of mouse-anti Ki67 antibody is substantially；Remain to colour developing during 1:800, but be obviously reduced；During 1:1500, antibody can not develop the color, therefore
1:1500 is the minimum working concentration of little mouse-anti Ki67 antibody；
(2) SABC method detection Ki67(A albumen):
A NSC creep plate that () cultivates, adds final concentration of 5 μm ol/L of BrdU() 37 DEG C hatch 4 hours after, 4%
Paraformaldehyde is fixed 20 minutes, with 0.01 mol/L PBS three times, each 5 minutes；
B () adds Normal Goat Serum confining liquid, hatch 30 minutes in 37 DEG C of wet boxes；
C () is pressed 1:1500 with antibody diluent and is diluted little mouse-anti Ki67 antibody, join cell climbing sheet, and 4 DEG C to hatch 24 little
Time；
D () PBS three times, after each 5 minutes, adds the anti-mouse IgG-by the dilution of proper proportion antibody diluent
Biotin, incubated at room 3 hours；
E () PBS three times, after each 5 minutes, adds fluorescein-labeled Streptavidin (SA-Cy2), room temperature is kept away
Light hatches 2 hours；
(f) PBS twice, fluorescence microscopy, observe coloration result；
(3) indirect method detection BrdU(B antigen):
A cell climbing sheet that () PBS Ki67 has developed the color, each 5 minutes, adds by proper proportion antibody diluent dilute
The little mouse-anti BrdU antibody (Sigma company, article No. B-8434) released, hatches 24 hours for 4 DEG C；
(b) PBS three times, after each 5 minutes, anti-mouse IgG-TRITC that addition dilutes with antibody diluent, room temperature
Lucifuge hatches 2 hours；
(c) PBS twice, 80% glycerol buffer mounting, fluorescence microscopy；
(4) fluorescence picture overlay analysis:
A () fluorescence microscope is taken pictures, obtain little mouse-anti Ki67 antibody and the stained photographs of little mouse-anti BrdU antibody；
B () utilizes software to be overlapped photo, result as shown in Figure 2: Fig. 2 a Green fluorescence represents purpose A albumen
(Ki67), Fig. 2 b red fluorescence represents purpose B antigen (BrdU), and Fig. 2 c is the superposition picture of the two, and the cell shown in arrow is altogether
Expressing K i67 and BrdU, cell expressing K i67 shown in asterisk.
Claims (2)
1. a Double-immunofluorescent labeling method based on same Species origin first antibody, it is characterised in that: same with two kinds
It is double that the anti-A protein antibodies of Species origin and anti-B protein antibodies carry out immunofluorescence to the A albumen in sample to be detected and B albumen
Heavy label；Utilize SABC immunofluorescence technique and IIF, be same at anti-A protein antibodies and anti-B protein antibodies
When belonging to source, A albumen or B albumen to cell or tissue carry out double labeling detection；
Comprise the following steps:
(1) anti-A protein antibodies minimum working concentration is determined；
(2) SABC method detection A albumen；
(3) indirect method detection B albumen；
(4) fluorescence picture overlay analysis.
Double-immunofluorescent labeling method the most according to claim 1, it is characterised in that comprise the following steps:
(1) anti-A protein antibodies minimum working concentration is determined
A () prepares histotomy to be measured or cell climbing sheet；
B () serum is closed；
C () hatches two times of gradient dilution anti-A protein antibodies；
D () is hatched the fluorescein for anti-A protein antibodies Species origin and is marked SA；
E () fluorescence microscopy, analyzes staining conditions, find out anti-A protein antibodies and just can not detect that A albumen to be measured is
Little working concentration；
(2) SABC method detection A albumen
A () prepares histotomy or cell climbing sheet according to step (1)；
B () serum is closed；
C () uses step (1) to obtain minimum working concentration and hatches anti-A protein antibodies；
D () hatches the biotin labeled SA for antibody A Species origin；
E () hatches fluorescein-labeled Streptavidin；
F () fluorescence microscopy, observes staining conditions；
(3) indirect method detection B albumen
A () utilizes the histotomy or cell climbing sheet that step (2) developed the color, hatch anti-B protein antibodies；
B () hatches fluorescein mark SA；
C () fluorescence microscopy, analyzes staining conditions；
(4) fluorescence picture overlay analysis
(a) fluorescence microscopy, take pictures, obtain the microscopy photo of A and B albumen to be measured；
B () utilizes software to be overlapped photo, analyze the expression of two kinds of albumen.
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