CN105973681B - A kind of immunohistochemistry operating method - Google Patents
A kind of immunohistochemistry operating method Download PDFInfo
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- CN105973681B CN105973681B CN201610334601.5A CN201610334601A CN105973681B CN 105973681 B CN105973681 B CN 105973681B CN 201610334601 A CN201610334601 A CN 201610334601A CN 105973681 B CN105973681 B CN 105973681B
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- 238000011017 operating method Methods 0.000 title claims abstract description 28
- 238000003364 immunohistochemistry Methods 0.000 title claims abstract description 18
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000004043 dyeing Methods 0.000 claims abstract description 11
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 claims abstract description 10
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 102000016938 Catalase Human genes 0.000 claims abstract description 6
- 108010053835 Catalase Proteins 0.000 claims abstract description 6
- 239000000975 dye Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 20
- 235000019441 ethanol Nutrition 0.000 claims description 13
- 229960004756 ethanol Drugs 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 5
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 5
- GTDOPRQDTRLYAL-UHFFFAOYSA-N hydrogen peroxide;methanol Chemical compound OC.OO GTDOPRQDTRLYAL-UHFFFAOYSA-N 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 5
- 239000002699 waste material Substances 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 230000008859 change Effects 0.000 abstract description 2
- 238000010438 heat treatment Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000011126 aluminium potassium sulphate Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 description 1
- 229940050271 potassium alum Drugs 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011697 sodium iodate Substances 0.000 description 1
- 229940032753 sodium iodate Drugs 0.000 description 1
- 235000015281 sodium iodate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N1/44—Sample treatment involving radiation, e.g. heat
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention discloses a kind of immunohistochemistry operating method, 1) it is sliced, copies piece；2) dewaxing and aquation；3) antigen retrieval；4) endogenous catalase is closed；5) it dyes；6) it is anti-that I is added dropwise；7) it is anti-that II is added dropwise；8) it develops the color: using DAB color developing agent, develop the color at room temperature；9) mounting.The technical solution adopted in the present invention changes Conventional procedures and uses haematoxylin dyeing before dropwise addition I resists, II is anti-, resists conducive to dropwise addition I, the anti-rear antibody of II develops the color.Operation of the present invention step is simple, and only the time of the operation order to existing operating procedure and the reagent of use, operation is changed, and the change of existing operating effect can be realized, instill antibody after dyeing, convenient for holding the size and location of tissue.Operation of the present invention step is simple and can effectively solve some technical detail problems in experimental check analysis, is conducive to Experimental Standardization and operates, and can be avoided the waste of experiment reagent, has also reduced some errors in experiment to the greatest extent.
Description
Technical field
The present invention relates to laboratory inspection analysis methods, and in particular to a kind of immunohistochemistry operating method.
Background technique
When immunohistochemistry drop antibody is done in laboratory, discovery is incubated for box, buffer is all colourless, histotomy in repair process
Also antibody is dripped just without sense of direction, can not hold the size and location of antibody tissue without color afterwards, it can only be continuous when dripping antibody
Careful lookup, and increase the amount instilled.
During entirely drop antibody, the case where more drops, wrong drop, drip, will cause the waste of reagent, increase detection at
This, influences as a result, delaying the slice time.
It needs to look through during film-making, mostly drop reagent, to cause the increase of work step, the working time prolongs
Long, working efficiency reduces, and increases cost of labor indirectly.
Based on this, laboratory research staff has researched and developed a kind of immunohistochemistry operating method.
Summary of the invention
The technical problems to be solved by the present invention are: existing immunohistochemistry operating method in operation caused by try
Agent waste is more, and directionless sense during antibody is added dropwise, and can not hold the tissue size and location that antibody is added dropwise.Now provide
It is anti-to solve immunohistochemistry operation dropwise addition by adjusting the operating procedure of existing operating method for a kind of immunohistochemistry operating method
The technical problems such as when body, working efficiency is low, and quantity of solvent waste is big.
Of the invention is achieved through the following technical solutions:
A kind of immunohistochemistry operating method, including following operating procedure:
1) it is sliced, copies piece: histotomy being placed at room temperature and toasts 20min at 60min or 60 DEG C；
2) dewaxing and aquation: histotomy being placed in dimethylbenzene and is impregnated 10 minutes, impregnates 10 points again after replacing dimethylbenzene
Then clock successively impregnates 5 minutes in dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol respectively；
3) antigen retrieval: PH6.0 0.01M sodium citrate buffer solution is heated in electric furnace or water-bath to 95 DEG C, is put into
Histotomy heats 10-15 minutes；
4) it closes endogenous catalase: being impregnated 30 minutes using 0.3% hydrogen peroxide methanol；
5) it dyes: using haematoxylin dyeing 2 minutes；
6) it is anti-that I is added dropwise: it is 50 microlitres anti-to be added dropwise I, be stored at room temperature 1 hour or 4 DEG C overnight or 37 DEG C 1 hour；
7) it is anti-that II is added dropwise: it is 40-50 microlitres anti-to be added dropwise II, stand at room temperature or 37 DEG C at stand 1 hour；
8) it develops the color: using DAB color developing agent, develop the color at room temperature；
9) mounting.
Existing laboratory carries out the step of immunohistochemistry operating method and generally comprises slice, copy piece, is dewaxing, reparation, endogenous
Property enzyme blocks, drop I is anti-, drop II resists, develops the color, redying, mounting, and use the operating method that antibody is added dropwise, as a result histotomy is several
It is transparent, it is difficult to visually observe, and be difficult to hold the amount of the position and antibody when antibody is added dropwise, need using more reagents and
When spending more, while being easy to that mistake is added dropwise during whole operation.
Inventor carries out repetition test operation for existing operating method, and discovery uses technical solution of the present invention, changes
Operating procedure can solve a series of problems in the prior art.Increase between endogenous enzyme blocking operation and dropwise addition I resist, II is anti-
Staining procedure can eliminate the activity of endogenous enzyme, while, II anti-rear antibody colour developing anti-convenient for subsequent dropwise addition I, so that it is determined that
Histocyte endoantigen positions antigen, qualitative and quantitative study.
Further, it is successively adopted 6 minutes wash with distilled water after being impregnated 30 minutes in the step 4), PBS rinses 6 points
Clock.Main function is cleaned to soak, prevents from influencing subsequent experimental result.
Further, for the step 5) using after haematoxylin dyeing, haematoxylin formula is distilled water 1600ml, potassium alum
100g, hematoxylin 4.0, sodium iodate 0.8g, glycerol 400ml, are broken up using hydrochloride alcohol.Haematoxylin liquid will be heated to when dyeing
It 40 degree-45 degree, contaminates 10-15 minutes, can get preferable experiment effect.
Further, the tween-20 of the anti-middle addition 0.05% of the step 7) II.Main function is that elution is non-specific
Absorption, under the influence of preventing in an operating procedure antibody colour developing.
Further, it includes containing 3%BSA and 10% serum that I is anti-in the step 6).
Further, the rewarming 37 DEG C at is added after I resists in the step 6).
Further, the step 7) is added after II resists and is rinsed 6 minutes with PBS.
Further, it is developed the color at room temperature 3-10 minutes in the step 8) using DBA color developing agent.
Further, the step 9) concrete operation method be by neutral gum drop beside the tissue of histotomy, then
It is covered with cleaning coverslip, is first laid flat side, then gently puts down the other side, sealed piece and be placed in vent cabinet and dry.
Compared with prior art, the present invention having the following advantages and benefits:
(1) the technical solution adopted in the present invention changes Conventional procedures before dropwise addition I resists, II is anti-using bush
Uniformly dyeing color, conducive to being added dropwise, I is anti-, the anti-rear antibody of II develops the color.
(2) operation of the present invention step is simple, only the operation order to existing operating procedure and the reagent of use, operation when
Between be changed, the change of existing operating effect can be realized, instill antibody after dyeing, convenient for hold tissue size and position
It sets.
(3) the configuration of the present invention is simple, it is easy to operate, and can effectively solve that some technical details are asked in experimental check analysis
Topic is conducive to Experimental Standardization and operates, has stronger practical application value, and can be avoided the waste of experiment reagent, also subtract as far as possible
Some errors in experiment are lacked.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment, the present invention is made
Further to be described in detail, exemplary embodiment of the invention and its explanation for explaining only the invention, are not intended as to this
The restriction of invention.
Embodiment 1:
A kind of immunohistochemistry operating method, including following operating procedure: 1) it is sliced, copies piece: at room temperature by histotomy
Place 60min；
2) dewaxing and aquation: histotomy being placed in dimethylbenzene and is impregnated 10 minutes, impregnates 10 points again after replacing dimethylbenzene
Then clock successively impregnates 5 minutes in dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol respectively；
3) antigen retrieval: using electric furnace heating pH is 6.0 0.01M sodium citrate buffer solution to 95 DEG C, is put into tissue
Slice heating 10 minutes；
4) it closes endogenous catalase: being impregnated 30 minutes using 0.3% hydrogen peroxide methanol；
5) it dyes: using haematoxylin dyeing 2 minutes；
6) it is anti-that I is added dropwise: I is added dropwise and resists 50 microlitres, is stored at room temperature 1 hour；
7) it is anti-that II is added dropwise: II is added dropwise and resists 40-50 microlitres, stands at room temperature；
8) it develops the color: using DAB color developing agent, develop the color at room temperature；
9) mounting.
Embodiment 2:
A kind of immunohistochemistry operating method, including following operating procedure: 1) it is sliced, copies piece: by histotomy at 60 DEG C
Toast 20min；；
2) dewaxing and aquation: histotomy being placed in dimethylbenzene and is impregnated 10 minutes, impregnates 10 points again after replacing dimethylbenzene
Then clock successively impregnates 5 minutes in dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol respectively；
3) antigen retrieval: heating pH6.0 0.01M sodium citrate buffer solution is put into histotomy to 95 DEG C in water-bath
Heating 15 minutes；
4) it closes endogenous catalase: being impregnated 30 minutes using 0.3% hydrogen peroxide methanol, then successively using steaming
Distilled water is cleaned 6 minutes, and PBS is rinsed 6 minutes；
5) it dyes: being broken up using after haematoxylin dyeing 2 minutes with hydrochloride alcohol；
6) it is anti-that I is added dropwise: it is 50 microlitres anti-to be added dropwise I, be stored at room temperature 1 hour or 4 DEG C overnight or 37 DEG C 1 hour；
7) it is anti-that II is added dropwise: it is 50 microlitres anti-to be added dropwise II, 37 DEG C 1 hour；
The wherein tween-20 of the anti-middle addition 0.05% of II.
8) it develops the color: using DAB color developing agent, develop the color at room temperature；
9) mounting: neutral gum drop is covered beside the tissue of histotomy, then with cleaning coverslip, is first laid flat one
Then the other side is gently put down in side, seal piece and be placed in vent cabinet and dry.
Embodiment 3:
A kind of immunohistochemistry operating method, including following operating procedure:
1) it is sliced, copies piece: histotomy is placed into 60min at room temperature；
2) dewaxing and aquation: histotomy being placed in dimethylbenzene and is impregnated 10 minutes, impregnates 10 points again after replacing dimethylbenzene
Then clock successively impregnates 5 minutes in dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol respectively；
3) antigen retrieval: it is 6.0 0.01M sodium citrate buffer solution to 95 DEG C that pH is heated in water-bath, is put into group
Knit slice heating 12 minutes；
4) it closes endogenous catalase: being impregnated 30 minutes using 0.3% hydrogen peroxide methanol；
5) it dyes: using haematoxylin dyeing 2 minutes；
6) it is anti-that I is added dropwise: standing 1 hour at dropwise addition I is 50 microlitres, 37 DEG C anti-；Wherein, I anti-ingredient is to include containing 3%BSA
With 10% serum
7) it is anti-that II is added dropwise: standing 1 hour at dropwise addition II is 40-50 microlitres, 37 DEG C anti-, is then rinsed 6 minutes with PBS；
8) it develops the color: using DAB color developing agent, develop the color 3-10 minutes at room temperature；
9) mounting.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (4)
1. a kind of immunohistochemistry operating method, which comprises the following steps:
1) it is sliced, copies piece: histotomy being placed at room temperature and toasts 20min at 60min or 60 DEG C；
2) dewaxing and aquation: histotomy being placed in dimethylbenzene and is impregnated 10 minutes, impregnates 10 minutes again after replacement dimethylbenzene, so
It is successively impregnated respectively 5 minutes in dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol afterwards；
3) antigen retrieval: it is 6.0 0.01M sodium citrate buffer solution to 95 DEG C that pH is heated in electric furnace or water-bath, is put into
Histotomy heats 10-15 minutes；
4) it closes endogenous catalase: being impregnated 30 minutes using 0.3% hydrogen peroxide methanol；
5) it dyes: using haematoxylin dyeing 2 minutes；
6) it is anti-that I is added dropwise: it is 50 microlitres anti-to be added dropwise I, be stored at room temperature 1 hour or 4 DEG C overnight or 37 DEG C 1 hour；
7) it is anti-that II is added dropwise: it is 40-50 microlitres anti-to be added dropwise II, stand at room temperature or 37 DEG C 1 hour；
8) it develops the color: using DAB color developing agent, develop the color at room temperature；
9) mounting；
Wherein, it is successively adopted 6 minutes wash with distilled water after being impregnated 30 minutes in the step 4), PBS is rinsed 6 minutes；
Wherein, then the step 5) is broken up using haematoxylin with hydrochloride alcohol；
Wherein, the tween-20 of the anti-middle addition 0.05% of the step 7) II.
2. a kind of immunohistochemistry operating method according to claim 1, it is characterised in that: after step 7) the addition II is anti-
It is rinsed 6 minutes with PBS.
3. a kind of immunohistochemistry operating method according to claim 1, it is characterised in that: use DBA in the step 8)
Color developing agent develops the color 3-10 minutes at room temperature.
4. a kind of immunohistochemistry operating method according to claim 1, it is characterised in that: the step 9) concrete operations side
Method is to cover neutral gum drop beside the tissue of histotomy, then with cleaning coverslip, is first laid flat side, then gently puts
The lower other side, seals piece and is placed in vent cabinet and dry.
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| CN107367607A (en) * | 2017-05-25 | 2017-11-21 | 长沙金域医学检验所有限公司 | A kind of Pathologic specimen section SABC operating method and its system |
| CN109669040B (en) * | 2018-12-14 | 2022-03-04 | 厦门艾德生物医药科技股份有限公司 | Antibody diluent for enhancing use effect of PD-L1 monoclonal antibody and use method thereof |
| CN110346552A (en) * | 2019-07-25 | 2019-10-18 | 深圳褀氏生物医疗电子有限公司 | A kind of universal antigen retrieval buffer |
| CN112485426A (en) * | 2020-12-01 | 2021-03-12 | 山东省药物研究院 | Circulating tumor cell IHC antigen repairing method based on ISET principle |
| CN112684178A (en) * | 2021-01-06 | 2021-04-20 | 深圳市圣通生物科技有限公司 | Immunohistochemical antigen repair buffer solution and use method thereof |
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| CN1963439A (en) * | 2006-11-27 | 2007-05-16 | 郑智勇 | Immunohistochemistry division bore dying paste, methods employed and purpose of the same |
| CN101726602A (en) * | 2009-12-11 | 2010-06-09 | 南开大学 | Method for judging ovarian cancer prognosis by detecting Legumain protein |
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| CN102707058B (en) * | 2012-05-30 | 2014-09-24 | 山东大学 | Tumor necrosis factor-alpha induced protein 8 L3 (TIPE3) immunohistochemistry detection kit for diagnosing lung cancer |
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|---|---|---|---|---|
| CN1963439A (en) * | 2006-11-27 | 2007-05-16 | 郑智勇 | Immunohistochemistry division bore dying paste, methods employed and purpose of the same |
| CN101726602A (en) * | 2009-12-11 | 2010-06-09 | 南开大学 | Method for judging ovarian cancer prognosis by detecting Legumain protein |
| CN102147417B (en) * | 2011-01-14 | 2013-11-06 | 中国农业大学 | Method for positioning immune tissues of growth hormone for malus plants and application thereof |
| CN102707058B (en) * | 2012-05-30 | 2014-09-24 | 山东大学 | Tumor necrosis factor-alpha induced protein 8 L3 (TIPE3) immunohistochemistry detection kit for diagnosing lung cancer |
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