CN105973681A - Immunohistochemical operation method - Google Patents

Immunohistochemical operation method Download PDF

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Publication number
CN105973681A
CN105973681A CN201610334601.5A CN201610334601A CN105973681A CN 105973681 A CN105973681 A CN 105973681A CN 201610334601 A CN201610334601 A CN 201610334601A CN 105973681 A CN105973681 A CN 105973681A
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minutes
sabc
antibody
operational approach
described step
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CN105973681B (en
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欧阳小峰
刘叶
谷和林
李军秀
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SICHUAN KINGMED DIAGNOSTICS CENTER CO Ltd
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SICHUAN KINGMED DIAGNOSTICS CENTER CO Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44Sample treatment involving radiation, e.g. heat
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77Systems 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
    • G01N21/78Systems 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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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • 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)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

The invention discloses an immunohistochemical operation method. The immunohistochemical operation method comprises the following steps of: (1) slicing and baking slices; (2) dewaxing and hydrating; (3) repairing an antigen; (4) sealing endogenous catalase; (5) dyeing; (6) dropwise adding an antibody I; (7) dropwise adding an antibody II; (8) developing, namely developing by adopting a DAB developing agent at a room temperature; and (9) sealing the slices. According to the technical scheme disclosed by the invention, a conventional operation step of dropwise adding the antibody I and the antibody II before dyeing by haematoxylin, the antibody can be easily developed after the antibody I and the antibody II are dropwise added. The immunohistochemical operation method is simple in operation steps; only an operation sequence of existing operation steps, adopted reagents and operation time are changed and the change of an existing operation effect can be realized; the antibodies are dropped after dyeing so that the size and position of tissues can be conveniently grasped. The operation steps are simple and certain technical detail problems in experiment check analysis can be effectively solved; standard operation of an experiment are easy to realize, wastes of experiment reagents can be avoided and certain errors in the experiment are also reduced as much as possible.

Description

A kind of SABC operational approach
Technical field
The present invention relates to laboratory inspection and analyze method, be specifically related to a kind of SABC operational approach.
Background technology
Laboratory does SABC when dripping antibody, it is all that colourless, tissue slice does not has color after repair process yet that box, buffer are hatched in discovery, and dripping antibody does not just have sense of direction, it is impossible to hold the size and location of antibody tissue, drip antibody time can only be the most careful lookup, and increase instill amount.
During whole antibody, many, wrong drip, the situation of drip can cause the waste of reagent, increase testing cost, affect result, delay the slice time.
Needing during film-making to look through, many reagent, thus cause the increase of job step, the working time extends, and work efficiency reduces, and indirectly adds cost of labor.
Based on this, laboratory research staff has researched and developed a kind of SABC operational approach.
Summary of the invention
The technical problem to be solved is: the reagent waste that existing SABC operational approach causes in operation is more, and directionless sense during dropping antibody, it is impossible to hold the tissue size and location of dropping antibody.A kind of SABC operational approach is now provided, by adjusting the operating procedure of existing operational approach, when solving SABC operation dropping antibody, inefficiency, the technical problems such as quantity of solvent waste is big.
The present invention is achieved through the following technical solutions:
A kind of SABC operational approach, including following operating procedure:.
1) cut into slices, copy sheet: tissue slice is at room temperature placed and at 60min or 60 DEG C, toasts 20min;;
2) dewaxing and aquation: tissue slice is placed in dimethylbenzene immersion 10 minutes, soaks 10 minutes after changing dimethylbenzene again, soaks 5 minutes respectively the most successively in dehydrated alcohol, 95% ethanol, 70% ethanol;
3) antigen retrieval: heating PH6.0 0.01M sodium citrate buffer solution is to 95 DEG C in electric furnace or water-bath, puts into tissue slice and heats 10 15 minutes;
4) endogenous catalase is closed: use 0.3% hydrogen peroxide methanol to soak 30 minutes;
5) dyeing: use haematoxylin dyeing 2 minutes;
6) dropping I resist: dropping the anti-50ul of I, room temperature stand 1 hour or 4 DEG C overnight or 37 DEG C 1 hour;
7) dropping II resists: dropping II resists 40 50ul, stands 1 hour at left at room temperature or 37 DEG C;
8) colour developing: use DAB developer, develops the color under room temperature;
9) mounting.
Existing laboratory carry out the step of SABC operational approach generally comprise section, copy sheet, dewax, repair, endogenous enzyme blocks, it is anti-to drip I, drip an II resists, develop the color, redye, mounting, and use this operational approach to drip antibody, result tissue slice is nearly transparent, it is difficult to perusal, and be difficult to hold position when dripping antibody and the amount of antibody, when needing use more reagent and spend more, it is easy to drip mistake in whole operating process simultaneously.
Inventor carries out repetition test operation for existing operational approach, finds to use technical solution of the present invention, changes operating procedure and can solve series of problems in prior art.Increase staining procedure between, II anti-in endogenous enzyme blocking-up operation and dropping I is anti-, the activity of endogenous enzyme can be eliminated, simultaneously facilitate that follow-up dropping I is anti-, II anti-after antibody colour developing, so that it is determined that histiocyte endoantigen, antigen is positioned, qualitative and quantitative study.
Further, using distilled water to clean in described step 4) after soaking 30 minutes successively 6 minutes, PBS rinses 6 minutes.Main Function is to be carried out soak, prevents the experimental result affected below.
Further, described step 5) use after haematoxylin dyeing, haematoxylin formula is distilled water 1600ml, potassium alum 100g, hematoxylin 4.0, sodium iodate 0.8g, glycerol 400ml, uses hydrochloride alcohol differentiation.Haematoxylin liquid will be heated to 40 degree 45 degree during dyeing, and contaminate 10 15 minutes, preferably experiment effect can be obtained.
Further, the tween-20 of 0.05% is added during described step 7) II is anti-.Main Function is eluting non-specific adsorption, prevents from affecting the colour developing of antibody in next operating procedure.
Further, in described step 6), I is anti-includes containing 3%BSA and 10% serum.
Further, described step 6) adds I anti-after at 37 DEG C rewarming.
Further, described step 7) adds after II resists with PBS rinsing 6 minutes.
Further, described step 8) use DBA developer at room temperature develop the color 3-10 minute..
Further, described step 9) concrete operation method is the tissue side that neutral gum drops in tissue slice, then with on cleaning coverslip lid, first sets level side, put down opposite side the most gently, seals sheet and is placed in ventilated chamber and dries.
The present invention compared with prior art, has such advantages as and beneficial effect:
(1) the technical solution adopted in the present invention change that Conventional procedures is anti-at dropping I, II anti-before use haematoxylin dyeing, be beneficial to that dropping I is anti-, II anti-after antibody colour developing.
(2) operating procedure of the present invention is simple, is only changed the operation order of existing operating procedure and the reagent, the time of operation that use, can realize the change of existing operating effect, instill antibody, it is simple to hold the size and location of tissue after dyeing.
(3) present configuration is simple, easy to operate, and can effectively solve the problem that some ins and outs problems in experimental check analysis, it is beneficial to Experimental Standardization operation, there is stronger actual application value, and it can be avoided that the waste of experiment reagent, decrease some errors in experiment the most as far as possible.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is described in further detail, and the exemplary embodiment of the present invention and explanation thereof are only used for explaining the present invention, not as a limitation of the invention.
Embodiment 1:
A kind of SABC operational approach, including following operating procedure: 1) cut into slices, copy sheet: tissue slice is at room temperature placed 60min;
2) dewaxing and aquation: tissue slice is placed in dimethylbenzene immersion 10 minutes, soaks 10 minutes after changing dimethylbenzene again, soaks 5 minutes respectively the most successively in dehydrated alcohol, 95% ethanol, 70% ethanol;
3) antigen retrieval: use heating by electric cooker PH be the 0.01M sodium citrate buffer solution of 6.0 to 95 DEG C, put into tissue slice heat 10 minutes;
4) endogenous catalase is closed: use 0.3% hydrogen peroxide methanol to soak 30 minutes;
5) dyeing: use haematoxylin dyeing 2 minutes;
6) dropping I resists: the dropping anti-50ul of I, and room temperature stands 1 hour;
7) dropping II resists: dropping II resists 40 50ul, left at room temperature;
8) colour developing: use DAB developer, develops the color under room temperature;
9) mounting.
Embodiment 2:
A kind of SABC operational approach, including following operating procedure: 1) cut into slices, copy sheet: tissue slice is toasted at 60 DEG C 20min;;
2) dewaxing and aquation: tissue slice is placed in dimethylbenzene immersion 10 minutes, soaks 10 minutes after changing dimethylbenzene again, soaks 5 minutes respectively the most successively in dehydrated alcohol, 95% ethanol, 70% ethanol;
3) antigen retrieval: in water-bath, heating PH6.0 0.01M sodium citrate buffer solution is to 95 DEG C, puts into tissue slice and heats 15 minutes;
4) endogenous catalase is closed: using 0.3% hydrogen peroxide methanol to soak 30 minutes, use distilled water to clean the most successively 6 minutes, PBS rinses 6 minutes;
5) dyeing: use haematoxylin dyeing to break up with hydrochloride alcohol after 2 minutes;
6) dropping I resist: dropping the anti-50ul of I, room temperature stand 1 hour or 4 DEG C overnight or 37 DEG C 1 hour;
7) dropping II resist: dropping the anti-50ul of II, 37 DEG C 1 hour;
The tween-20 of 0.05% is added during wherein II is anti-.
8) colour developing: use DAB developer, develops the color under room temperature;
9) mounting: neutral gum drops in the tissue side of tissue slice, then with on cleaning coverslip lid, first set level side, put down opposite side the most gently, seals sheet and is placed in ventilated chamber and dries.
Embodiment 3:
A kind of SABC operational approach, including following operating procedure:
1) cut into slices, copy sheet: tissue slice is at room temperature placed 60min;
2) dewaxing and aquation: tissue slice is placed in dimethylbenzene immersion 10 minutes, soaks 10 minutes after changing dimethylbenzene again, soaks 5 minutes respectively the most successively in dehydrated alcohol, 95% ethanol, 70% ethanol;
3) antigen retrieval: in water-bath heat PH be the 0.01M sodium citrate buffer solution of 6.0 to 95 DEG C, put into tissue slice heat 12 minutes;
4) endogenous catalase is closed: use 0.3% hydrogen peroxide methanol to soak 30 minutes;
5) dyeing: use haematoxylin dyeing 2 minutes;
6) dropping I resists: the dropping anti-50ul of I, stands 1 hour at 37 DEG C;Wherein, anti-for I composition is for including containing 3%BSA and 10% serum
7) dropping II resists: dropping II resists 40 50ul, stands 1 hour at 37 DEG C, then rinses 6 minutes with PBS;
8) colour developing: use DAB developer, develops the color 3-10 minute under room temperature;
9) mounting.
Above-described detailed description of the invention; the purpose of the present invention, technical scheme and beneficial effect are further described; it is it should be understood that; the foregoing is only the detailed description of the invention of the present invention; the protection domain being not intended to limit the present invention; all within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. done, should be included within the scope of the present invention.

Claims (9)

1. a SABC operational approach, it is characterised in that comprise the following steps:
1) cut into slices, copy sheet: tissue slice is at room temperature placed and at 60min or 60 DEG C, toasts 20min;
2) dewaxing and aquation: tissue slice is placed in dimethylbenzene immersion 10 minutes, soaks 10 minutes after changing dimethylbenzene again, soaks 5 minutes respectively the most successively in dehydrated alcohol, 95% ethanol, 70% ethanol;
3) antigen retrieval: in electric furnace or water-bath heat PH be the 0.01M sodium citrate buffer solution of 6.0 to 95 DEG C, put into tissue slice and heat 10 15 minutes;
4) endogenous catalase is closed: use 0.3% hydrogen peroxide methanol to soak 30 minutes;
5) dyeing: use haematoxylin dyeing 2 minutes;
6) dropping I resist: dropping the anti-50ul of I, room temperature stand 1 hour or 4 DEG C overnight or 37 DEG C 1 hour;
7) dropping II resists: dropping II resists 40 50ul, left at room temperature or 37 DEG C 1 hour;
8) colour developing: use DAB developer, develops the color under room temperature;
9) mounting.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: using distilled water to clean after soaking 30 minutes in described step 4) successively 6 minutes, PBS rinses 6 minutes.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: described step 5) uses haematoxylin then to break up with hydrochloride alcohol.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: add the tween-20 of 0.05% during described step 7) II is anti-.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: in described step 6), I is anti-includes containing 3%BSA and 10% serum.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: described step 6) adds I anti-after at 37 DEG C rewarming.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: described step 7) adds after II resists with PBS rinsing 6 minutes.
A kind of SABC operational approach the most according to claim 1, it is characterised in that: described step 8) use DBA developer at room temperature develop the color 3-10 minute.
A kind of SABC operational approach the most according to claim 1, it is characterized in that: described step 9) concrete operation method is the tissue side that neutral gum drops in tissue slice, again with on cleaning coverslip lid, first set level side, put down opposite side the most gently, seal sheet and be placed in ventilated chamber and dry.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109669040A (en) * 2018-12-14 2019-04-23 厦门艾德生物医药科技股份有限公司 It is a kind of for enhancing the antibody diluent and its application method of PD-L1 monoclonal antibody using effect
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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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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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109669040A (en) * 2018-12-14 2019-04-23 厦门艾德生物医药科技股份有限公司 It is a kind of for enhancing the antibody diluent and its application method of PD-L1 monoclonal antibody using effect
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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