CN108680419A - A kind of secondary antibody colouring method for immunohistochemistry autostainer - Google Patents
A kind of secondary antibody colouring method for immunohistochemistry autostainer Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The present invention relates to a kind of secondary antibody colouring method more particularly to a kind of secondary antibody colouring methods for immunohistochemistry autostainer.This approach includes the following steps：S1, it is added in histotomy and blockades liquid, make endogenic peroxidase inactivation in histotomy cell；S2, corresponding primary antibody is added in histotomy, the antigen protein reaction bonded intracellular with histotomy；S3, increased response liquid is added in histotomy；S4, polymer secondary antibody, and the combination of primary antibody specificity are added in histotomy；S5, the mixed liquor that DAB concentrates and DAB buffer are added in histotomy, DAB precipitation colorings；S6, addition haematoxylin redyes liquid in histotomy, and aobvious blue is combined with chromatin.Present invention optimizes agent formulations needed for the experiment of routine immunization groupization, improve the staining power of immunohistochemistry, reduce issuable unspecific staining background, greatly improve dyeing quality.
Description
Technical field
The present invention relates to a kind of secondary antibody colouring method more particularly to a kind of secondary antibodies for immunohistochemistry autostainer
Colouring method.
Background technology
In Clinicopathologic Diagnosis and Senile Mouse, immunohistochemistry (abbreviation immunohistochemistry) dyeing is a kind of very heavy
The technology and means wanted.Immunohistochemistry technique in the seventies in last century applied to pathological diagnosis begin, at present global pathology circle
Through being used widely, it has become indispensable part in pathologist routine work.Immunohistochemistry technique possesses specifically
Property, the advantages that sensibility is strong and easy to operate so that immunohistochemistry technique medical diagnosis on disease field obtained extensive popularization and
Using especially Clinicopathologic Diagnosis and tumor transformation diagnose.Immunohistochemistry technique can not only improve the accuracy of pathological diagnosis,
Clinical and basic subject has also been penetrated into simultaneously, has been played not in the teiology, pathogenesis and research work for inquiring into disease
Appreciable effect.
Immunohistochemistry is the specific binding using antigen-antibody, by the colour developing of marker, to detect and position tissue
Or the antigen protein in cell.Plurality of reagents is needed to be used cooperatively in immunohistochemical experiment, conventional immunohistochemical staining is logical
It is often to be carried out by manual operations, process is cumbersome time-consuming and long, and stability is poor, and repeatability is relatively low.Used in hand dyeing
Sensitivity, compatibility, stability and the quality of reagent are all insufficient, and it is irregular that this also results in hand dyeing quality, very
Difficulty fully meets the requirement of Clinicopathologic Diagnosis.And the appearance of immunohistochemistry autostainer greatly improves dyeing effect
Rate is not necessarily to manual intervention, simple operation in operational process.But general immunohistochemistry reagent is only applicable to manually operated dye
Color requirement, and immunohistochemistry autostainer can not be applied to so that dyeing sensitivity is relatively low.
Invention content
The present invention in order to solve the above technical problems, provide reasonable design, significantly improve one kind of coloring for it is complete from
The secondary antibody colouring method of dynamic immunohistochemical stainer.
Technical solution is used by the present invention solves above-mentioned technical problem：
A kind of secondary antibody colouring method for immunohistochemistry autostainer includes the following steps：
S1, it is added in histotomy and blockades liquid, make endogenic peroxidase inactivation in histotomy cell；
S2, corresponding primary antibody is added in histotomy, the antigen protein reaction bonded intracellular with histotomy；
S3, increased response liquid is added in histotomy；
S4, polymer secondary antibody, and the combination of primary antibody specificity are added in histotomy；
S5, the mixed liquor that DAB concentrates and DAB buffer are added in histotomy, DAB precipitation colorings；
S6, addition haematoxylin redyes liquid in histotomy, and aobvious blue is combined with chromatin.
Present invention optimizes agent formulations, the dyeing for improving immunohistochemistry needed for the experiment of routine immunization groupization are strong
Degree, reduces issuable unspecific staining background, greatly improves dyeing quality.
Further, the liquid of blockading is hydrogenperoxide steam generator, a concentration of 3%-4% of the hydrogenperoxide steam generator.
Further, the increased response liquid is rabbit anti-mouse igg solution, a concentration of 5~10 μ of the rabbit anti-mouse igg solution
g/mL。
Further, the DAB concentrates are 3,3- diaminobenzidines, four hydrogen chloride solution, 3, the 3- benzidines
A concentration of 55~70mmol/L of four hydrogen chloride solution of amine.
Further, the DAB Buffer are 0.05~0.15% hydrogenperoxide steam generator.
Further, the haematoxylin redyes a concentration of the 0.05~0.15% of liquid.
Further, further include the making of step S0 tissue section slides before step S1, step is：
Step 1: roasting piece, dries the moisture on slide；
Step 2: dewaxing, dissolves the paraffin of investing tissue so that histotomy is completely exposed；
Step 3: aquation, alcohol washes away dewaxing liquid, and histotomy is made to be full of water into the cell；
Step 4: antigen retrieval, is added antigen retrieval buffers, exposure antigenic determinant in histotomy.
Further, further include the processing of step S7 dyeing glass slides after step S6, include the following steps：
Step 1: dehydration, is successively separately added into 80% alcohol, 90% alcohol, 95% alcohol and 100% on dyeing glass slide
Alcohol carries out serial dehydration；
Step 2: it is transparent, dewaxing liquid is added on dyeing glass slide；
Step 3: mounting, is added neutral gum progress mounting on dyeing glass slide.
The present invention compared with the existing technology has the following advantages and effect：
1, present invention optimizes agent formulations, the dyeing for improving immunohistochemistry needed for the experiment of routine immunization groupization are strong
Degree, reduces issuable unspecific staining background, greatly improves dyeing quality；
2, it present invention comprises the necessary reagent of immunohistochemical staining, matches in immunohistochemistry autostainer,
With high sensitivity and affinity；Endogenic peroxidase and nonspecific protein binding site can be closed,
Reduce dyeing background that may be present, so that background coloration is more clear clean free from admixture noresidue, solve shadow for a long time
The background coloration problem for ringing dyeing quality can fully meet the pathological diagnosis requirement of instrument dyeing；
3, the present invention is apparent in the instrument coloring of different tissues different antibodies.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without having to pay creative labor, may be used also for those of ordinary skill in the art
With obtain other attached drawings according to these attached drawings.
Fig. 1 is the structural schematic diagram of reagent apparatus for placing of the present invention.
Label declaration：
1, reagent bottle, 3, reagent bottle label.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, following embodiment be explanation of the invention and
The invention is not limited in following embodiments.
Immunohistochemistry autostainer in the present invention belongs to the prior art, which is applicable in
In a variety of samples such as paraffin, frost, puncture, cell smear, marrow pieces；It is soft that control is provided on immunohistochemistry autostainer
Part, may be programmed over one hundred kind of standardization immunohistochemistry and quick specific stain program, and program can be according to requiring to change at any time, customize
Characteristic suitable incubation time is arranged on immunohistochemistry autostainer, immunohistochemical staining can be completed.Automatically exempt from
The listing of epidemic disease histochemical staining instrument greatly improves the working efficiency of pathological diagnosis, is taken completely instead of very complicated interminable
Hand dyeing.
Separately there is reagent apparatus for placing to be used cooperatively with immunohistochemistry autostainer.
Embodiment 1：
A kind of secondary antibody colouring method for immunohistochemistry autostainer, including following methods：
S1, it is added in histotomy and blockades liquid 150ul, endogenic peroxidase in histotomy cell is made to lose
It is living；
S2, corresponding primary antibody 150ul is added in histotomy, the antigen protein intracellular with histotomy reacts knot
It closes；
S3, increased response liquid 150ul is added in histotomy；
S4, polymer secondary antibody 150ul, and the combination of primary antibody specificity are added in histotomy；
S5, addition is mixed by the DAB concentrates of 7.5ul and the DAB buffer of 150ul in histotomy
Mixed liquor, DAB precipitation colorings；
S6, addition haematoxylin redyes liquid 150ul in histotomy, and aobvious blue is combined with chromatin.
Another scheme, a kind of secondary antibody colouring method for immunohistochemistry autostainer, including following methods：
S1, it is added in histotomy and blockades liquid 100ul, endogenic peroxidase in histotomy cell is made to lose
It is living；
S2, corresponding primary antibody 100ul is added in histotomy, the antigen protein intracellular with histotomy reacts knot
It closes；
S3, increased response liquid 100ul is added in histotomy；
S4, polymer secondary antibody 100ul, and the combination of primary antibody specificity are added in histotomy；
S5, it is added in histotomy and is mixed by what the DAB concentrates of 5ul and the DAB buffer of 100ul were mixed
Close liquid, DAB precipitation colorings；
S6, addition haematoxylin redyes liquid 100ul in histotomy, and aobvious blue is combined with chromatin.
Chromatin is the carrier of inhereditary material.Chromatin refer in interphase nuclei by DNA, histone, it is nonhistones and
The linear composite construction of a small amount of RNA compositions, is form existing for Interphase cells inhereditary material.
In the present invention, blockade liquid for close the endogenic peroxidase of inactivation.
In the present invention, polymer secondary antibody is used for the primary antibody that the combination of specificity is reacted with antigen protein.
In the present invention, increased response liquid is used to enhance the binding ability of polymer secondary antibody and primary antibody, improves the knot of secondary antibody
Quantity is closed, the reaction sensitivity of antigen-antibody is increased, makes certain low-abundance protein stainings also can be ideal, color is more
Vivid, this solves certain immunohistochemistry reagent D AB and that colour cast is dark, and color is not bright-coloured enough, inclined for the protein staining of low expression
It is shallow, it is unable to reach ideal coloring.
In the present invention, colour developing of the DAB concentrates for the destination protein of HRP catalysis.DAB is Diaminobenzidine's
Abbreviation, Chinese is diaminobenzidine, is that horseradish peroxidase is most sensitive, most common chromogenic substrate, reaction product
It is widely used in western blot (Western Blot, WB) due to the brown precipitate of not soluble in water, dimethylbenzene and alcohol, is immunized
Histochemistry (Immunohistochemistry, IHC) and immunocytochemistry (Immunocytochemistry, ICC), spot
Put the dyeing and chromogenic reaction of trace (Dot blot) and biochip (Biochip) etc..Wherein HRP is horse radish
The abbreviation of peroxidase, Chinese horseradish peroxidase are a kind of peroxidase for studying most deep in plant.
In the present invention, DAB buffer are used to dilute the colour developing of DAB concentrates and the destination protein of HRP catalysis.
In the present invention, haematoxylin redyes liquid for chromatinic coloring, is by alum, hematoxylin, sodium iodate and ice
Acetic acid is add to deionized water, and is uniformly mixed and is formed.
Primary antibody is the abbreviation of first antibody, and being can be with the albumen of non-antibody antigen (specific antigen) specific binding.
It in the present invention, using mouse source or rabbit source primary antibody, and is contained in other containers, certainly by immunohistochemistry autostainer
It is dynamic to be added on histotomy.
As shown in Figure 1, being provided with 7 reagent bottles 1 on the reagent apparatus for placing 2, according to dyeing sequencing, i.e., will blockade
Liquid, increased response liquid, polymer secondary antibody, DAB concentrates, DAB buffer and haematoxylin redye liquid and are subsequently placed at examination according to this
In agent bottle 1, wherein DAB buffer usage amounts are big, so holding DAB buffer with two reagent bottles 1.Using full-automatic
When immunohistochemical stainer, immunohistochemistry autostainer scans 3 reading reagent bottle of reagent bottle label, 1 information, identification agent
The reagent information held in bottle 1, and carry out the absorption of reagent automatically and add reagent on slide.In full-automatic immunohistochemistry
In dyeing instrument operation, immunohistochemistry autostainer mixes DAB concentrates and DAB buffer automatically, without human intervention,
Avoid being in direct contact for carcinogen.
In step S1 in the present embodiment, after liquid is blockaded in histotomy addition, it is incubated at room temperature 10min, then with existing
TBS (Chinese is t-Butyldimethylsilyl) cleanings in technology are three times.
In the step S2 of the present embodiment, after primary antibody is added in histotomy, 30min is incubated in the environment of 37 degrees Celsius, so
It is cleaned three times with TBS afterwards.
In the step S3 of the present embodiment, after increased response liquid is added in histotomy, it is incubated at room temperature 15min, then TBS is clear
It washes three times.
In the step S4 of the present embodiment, the universal polymer secondary antibody of pika is added in histotomy, in 37 degrees Celsius of ring
20min is incubated under border, TBS is cleaned three times.
In the step S5 of the present embodiment, the DAB concentrates and DAB buffer mixed in advance is added in histotomy
Mixed liquor, room temperature dye 3-10min, and then deionized water is cleaned three times.Room temperature dyeing time is by the antigen with an anti-binding
Amount and the concentration of the mixed liquor of DAB concentrates and DAB buffer determine, belong to the prior art, herein not reinflated description.
In the step S6 of the present embodiment, histotomy be added haematoxylin redye liquid, room temperature redyes 30s, then spend from
Sub- water cleaning is three times.
Embodiment 2：
As described in Example 1, it is hydrogenperoxide steam generator to differ only in and blockade liquid, and hydrogenperoxide steam generator is a concentration of
3%.
Embodiment 3：
As described in Example 1, it is hydrogenperoxide steam generator to differ only in and blockade liquid, and hydrogenperoxide steam generator is a concentration of
3.5%.
Embodiment 4：
As described in Example 1, it is hydrogenperoxide steam generator to differ only in and blockade liquid, and hydrogenperoxide steam generator is a concentration of
4%.
Embodiment 5：
As described in Example 1, differ only in increased response liquid be rabbit anti-mouse igg solution, rabbit anti-mouse igg solution it is dense
Degree is 5 μ g/mL；Solution PH is 7.4.Wherein it is used for a concentration of the 0.05% of 950 solution of corrosion-resistant Proclin.
Certain protein (such as certain cell factor or memebrane protein) of mouse is foreign matter for rabbit, so working as mouse
This protein injection rabbit body in when, rabbit just will produce the antibody for the antigen, and certain antibody has several kinds, wherein
IgG is most important antibody.
Embodiment 6：
As described in Example 1, differ only in increased response liquid be rabbit anti-mouse igg solution, rabbit anti-mouse igg solution it is dense
Degree is 7.5 μ g/mL；Solution PH is 7.4.
Embodiment 7：
As described in Example 1, differ only in increased response liquid be rabbit anti-mouse igg solution, rabbit anti-mouse igg solution it is dense
Degree is 10 μ g/mL；Solution PH is 7.4.
Embodiment 8：
As described in Example 1, differ only in DAB concentrates be 3,3- diaminobenzidines, four hydrogen chloride solution, 3,
A concentration of 55mmol/L of four hydrogen chloride solution of 3- diaminobenzidines.
Embodiment 9：
As described in Example 1, differ only in DAB concentrates be 3,3- diaminobenzidines, four hydrogen chloride solution, 3,
A concentration of 62mmol/L of four hydrogen chloride solution of 3- diaminobenzidines.
Embodiment 10：
As described in Example 1, differ only in DAB concentrates be 3,3- diaminobenzidines, four hydrogen chloride solution, 3,
A concentration of 70mmol/L of four hydrogen chloride solution of 3- diaminobenzidines.
Embodiment 11：
As described in Example 1, the hydrogenperoxide steam generator that DAB Buffer are a concentration of 0.05% is differed only in.
Embodiment 12：
As described in Example 1, the hydrogenperoxide steam generator that DAB Buffer are a concentration of 0.10% is differed only in.
Embodiment 13：
As described in Example 1, the hydrogenperoxide steam generator that DAB Buffer are a concentration of 0.15% is differed only in.
Embodiment 14：
As described in Example 1, haematoxylin redyes liquid a concentration of 0.05% is differed only in.
Embodiment 15：
As described in Example 1, haematoxylin redyes liquid a concentration of 0.10% is differed only in.
Embodiment 16：
As described in Example 1, haematoxylin redyes liquid a concentration of 0.15% is differed only in.
Embodiment 17：
As shown in embodiment 1-16 any embodiments, it further includes that step S0 tissues are cut to differ only in before step S1
The making of piece slide, step are：
Step 1: roasting piece, dries the moisture on slide；
Step 2: dewaxing, the paraffin of investing tissue is dissolved with dewaxing liquid in the prior art so that histotomy is completely sudden and violent
Dew；
Step 3: aquation, alcohol washes away dewaxing liquid, and histotomy is made to be full of water into the cell；
Step 4: antigen retrieval, is added antigen retrieval buffers, exposure antigenic determinant in histotomy.
Antigen retrieval buffers can be that citric acid antigen in the prior art repairs liquid or EDTA antigen retrieval buffers.
Antigenic determinant can be made of or by discontinuous protein tridimensional continuous sequence (prlmary structure of protein)
Structure composition determines antigenic special chemical group, also known as epitope.Antigenic determinant is present in antigenic substance mostly
Surface, some are present in the inside of antigenic substance, must just be exposed after enzyme or other modes processing.One native antigen object
Matter can there are many and multiple determinants.Antigen molecule is bigger, and the number of determinant is more.
In the present embodiment, histotomy toasts 30min in 60 degrees Celsius of environment, dries the moisture on slide, makes
Histotomy is obtained to be adhering completely on glass slide.
In the present embodiment, dewaxing liquid is heated to 72 degrees Celsius and 2 dewaxings is carried out to histotomy, dissolve investing tissue
Paraffin so that histotomy is completely exposed.
In the present embodiment, it is added after antigen retrieval buffers in histotomy, is stood in the environment of 100 degrees Celsius
20min。
Embodiment 18：
As shown in embodiment 1-16 any embodiments, it further includes step S7 dyeing glass to differ only in after step S6
The processing of piece, includes the following steps：
Step 1: dehydration, is successively separately added into 80% alcohol, 90% alcohol, 95% alcohol and 100% on dyeing glass slide
Alcohol carries out serial dehydration；
Step 2: it is transparent, dewaxing liquid is added on dyeing glass slide；
Step 3: mounting, is added neutral gum progress mounting on dyeing glass slide.
Neutral gum is that a kind of adhesive seals biological tissue section for being bonded together glass slide and coverslip
Get up permanent reservation, belongs to the prior art.
In the present embodiment, slide is immersed in 80% alcohol 3 minutes when dehydration, is then immersed in 3 in 90% alcohol
Minute, it is then soaked in 95% alcohol 3 minutes, then be immersed in 100% alcohol 5 minutes, is finally immersed in 100% wine again
5 minutes in essence, serial dehydration is completed.
In the present embodiment, when carrying out transparent processing, dewaxing liquid impregnates 5 minutes, and then dewaxing liquid impregnates 5 points again
Clock.
In the present embodiment, after mounting, coloring is observed under the microscope and is taken pictures.
Furthermore, it is necessary to which explanation, is placed on according to prior art histotomy between slide and glass slide.This theory
Specific embodiment described in bright book, shape, named title of parts and components etc. can be different.It is all according to patent structure of the present invention
Think the equivalent or simple change that the structure, feature and principle is done, is included in the scope of protection of the invention patent.This
The technical staff of technical field that the present invention belongs to can make various modifications or additions to the described embodiments or adopt
It is substituted with similar mode, it, should all without departing from structure of the invention or beyond the scope defined by this claim
It belongs to the scope of protection of the present invention.
Claims (8)
1. a kind of secondary antibody colouring method for immunohistochemistry autostainer, it is characterised in that：Include the following steps：
S1, it is added in histotomy and blockades liquid, make endogenic peroxidase inactivation in histotomy cell；
S2, corresponding primary antibody is added in histotomy, the antigen protein reaction bonded intracellular with histotomy；
S3, increased response liquid is added in histotomy；
S4, polymer secondary antibody, and the combination of primary antibody specificity are added in histotomy；
S5, the mixed liquor that DAB concentrates and DAB buffer are added in histotomy, DAB precipitation colorings；
S6, addition haematoxylin redyes liquid in histotomy, and aobvious blue is combined with chromatin.
2. a kind of secondary antibody colouring method for immunohistochemistry autostainer according to claim 1, feature exist
In the liquid of blockading is hydrogenperoxide steam generator, a concentration of 3%-4% of the hydrogenperoxide steam generator.
3. a kind of secondary antibody colouring method for immunohistochemistry autostainer according to claim 1, feature exist
In the increased response liquid is rabbit anti-mouse igg solution, a concentration of 5~10 μ g/mL of the rabbit anti-mouse igg solution.
4. a kind of secondary antibody colouring method for immunohistochemistry autostainer according to claim 1, feature exist
In the DAB concentrates are 3,3- diaminobenzidines, four hydrogen chloride solution, and 3,3- diaminobenzidines, four hydrogen chloride is molten
A concentration of 55~70mmol/L of liquid.
5. a kind of secondary antibody colouring method for immunohistochemistry autostainer according to claim 1, feature exist
In the DAB Buffer are 0.05~0.15% hydrogenperoxide steam generator.
6. a kind of secondary antibody colouring method for immunohistochemistry autostainer according to claim 1, feature exist
In the haematoxylin redyes a concentration of the 0.05~0.15% of liquid.
7. a kind of two anti-dye for immunohistochemistry autostainer according to any claim from 1 to 6
Method, which is characterized in that further include the making of step S0 tissue section slides before step S1, step is：
Step 1: roasting piece, dries the moisture on slide；
Step 2: dewaxing, dissolves the paraffin of investing tissue so that histotomy is completely exposed；
Step 3: aquation, alcohol washes away dewaxing liquid, and histotomy is made to be full of water into the cell；
Step 4: antigen retrieval, is added antigen retrieval buffers, exposure antigenic determinant in histotomy.
8. a kind of two anti-dye for immunohistochemistry autostainer according to any claim from 1 to 6
Method, which is characterized in that further include the processing of step S7 dyeing glass slides after step S6, include the following steps：
Step 1: dehydration, is successively separately added into 80% alcohol, 90% alcohol, 95% alcohol and 100% wine on dyeing glass slide
Essence carries out serial dehydration；
Step 2: it is transparent, dewaxing liquid is added on dyeing glass slide；
Step 3: mounting, is added neutral gum progress mounting on dyeing glass slide.
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| CN104991073A (en) * | 2015-06-18 | 2015-10-21 | 南通大学附属医院 | Ready-to-use rapid enzyme immune tissue chemical reagent kit for detecting SCML2 |
| CN105699156A (en) * | 2016-02-24 | 2016-06-22 | 福州迈新生物技术开发有限公司 | Full-automatic staining instrument for staining tissue samples on glass slides and method for applying full-automatic staining instrument |
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| CN104991073A (en) * | 2015-06-18 | 2015-10-21 | 南通大学附属医院 | Ready-to-use rapid enzyme immune tissue chemical reagent kit for detecting SCML2 |
| CN105699156A (en) * | 2016-02-24 | 2016-06-22 | 福州迈新生物技术开发有限公司 | Full-automatic staining instrument for staining tissue samples on glass slides and method for applying full-automatic staining instrument |
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| CN111929432A (en) * | 2020-08-10 | 2020-11-13 | 英诺维尔智能科技（苏州）有限公司 | Operation method of high-throughput flexible tissue sample analysis system |
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Application publication date: 20181019 |