CN107367607A - A kind of Pathologic specimen section SABC operating method and its system - Google Patents
A kind of Pathologic specimen section SABC operating method and its system Download PDFInfo
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- CN107367607A CN107367607A CN201710380142.9A CN201710380142A CN107367607A CN 107367607 A CN107367607 A CN 107367607A CN 201710380142 A CN201710380142 A CN 201710380142A CN 107367607 A CN107367607 A CN 107367607A
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- 230000001575 pathological effect Effects 0.000 title claims abstract description 83
- 238000011017 operating method Methods 0.000 title claims abstract description 17
- 239000012188 paraffin wax Substances 0.000 claims abstract description 46
- 239000000427 antigen Substances 0.000 claims abstract description 19
- 102000036639 antigens Human genes 0.000 claims abstract description 19
- 108091007433 antigens Proteins 0.000 claims abstract description 19
- 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 15
- 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 15
- 238000000386 microscopy Methods 0.000 claims abstract description 14
- 238000004043 dyeing Methods 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- -1 DBA nitrite ions Chemical class 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 230000004069 differentiation Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims 1
- 229910052760 oxygen Inorganic materials 0.000 claims 1
- 239000001301 oxygen Substances 0.000 claims 1
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000001993 wax Substances 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
- 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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- Hematology (AREA)
- Urology & Nephrology (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
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Abstract
The invention discloses a kind of Pathologic specimen section SABC operating method and its system, the step such as mounting and microscopy that colour developing that dewaxing that operating method bakes piece including Pathologic specimen paraffin section, Pathologic specimen, Pathologic specimen is cut into slices, Pathologic specimen section antigen retrieval, Pathologic specimen section statining, Pathologic specimen are cut into slices and antibody are added dropwise, Pathologic specimen is cut into slices, Pathologic specimen section are redyed, Pathologic specimen is cut into slices.The present invention uses first carries out dyeing dropwise addition antibody I, antibody II with haematoxylin, beneficial to the colour developing of antibody, and the size and location that antibody is easy to hold tissue is instilled after dyeing, reduce the deviation held when antibody is added dropwise to position, it is easy to Experimental Standardization to operate, with stronger actual application value, actual waste can be effectively avoided, also can largely reduce the error of experimental data.
Description
Technical field
The invention belongs to the technical field of test in laboratory analysis method, more particularly to a kind of Pathologic specimen section immune group
Change operating method and its system.
Background technology
SABC is applied immunology antigen-antibody reaction principle, i.e. antigen and antibody specificity reaction principle, is passed through
Chemical reaction makes the developer such as colour developing such as fluorescein, enzyme, metal ion, isotope of labelled antibody to determine response organization's cell
Endoantigen, it is positioned, be qualitative and determine quantifier elimination.
And operated for SABC, experimenter does not have after frequently encountering histotomy repair process in operation
Color, be added dropwise I is anti-, II is anti-when, it is not known that the position being specifically added dropwise and tissue, large area can only be taken to be added dropwise, so as to
Cause the result that is added dropwise inaccurate, it is impossible to position, can not also carry out follow-up qualitative, quantitative to the antigen in histocyte
Research operation.
Therefore, an immunohistochemical experiment will pass through multi-pass operation, and experiment knot may be influenceed on whole experimental implementation
The link of structure is excluded one by one, and workload is big, greatly reduces operating efficiency.Based on this, laboratory research staff is more
A kind of SABC operating method of New tissue section is devised in the practice operation of secondary problem.
The content of the invention
The present invention solves operating personnel of the prior art position when antibody is added dropwise when carrying out immunohistochemical experiment
Hold inaccurate, sick operating efficiency is low, and waste of solvent is big, the problems such as experimental data inaccuracy.
In order to solve the above-mentioned technical problem, the invention provides a kind of Pathologic specimen section SABC operating method, bag
Include following steps：
(1) Pathologic specimen paraffin section：Produced Pathologic specimen paraffin is taken, 2-4 μm of stone is cut into paraffin slicing machine
Wax is cut into slices；
(2) Pathologic specimen bakes piece：The slide 2h with Pathologic specimen paraffin section is baked in 55-65 DEG C of insulating box；
(3) Pathologic specimen section dewaxing：First the slide with Pathologic specimen paraffin section is put at room temperature before dewaxing
Put 60min, soaked afterwards with dimethylbenzene I, dimethylbenzene II, dimethylbenzene III, each 5min, then by slide be placed on absolute alcohol I,
Soaked in absolute alcohol II, absolute alcohol III, each 2min, then move to 95% alcohol I, 95% alcohol II, 85% alcohol successively
Middle immersion, each 2min, finally rinse 2min with PBS；
(4) Pathologic specimen section antigen retrieval：By the slide after rinsing well be put into PH in pressure cooker be 8.0 it is anti-
Original repairs 20-25min in liquid, and more than natural cooling 30min, PBS are rinsed 2-3 times, then moved to and 2min is soaked in distilled water,
8min is soaked in 3% hydrogen peroxide, PBS is rinsed 2-3 times；
(5) Pathologic specimen section statining：Using haematoxylin dyeing 2-3min；
(6) antibody is added dropwise in Pathologic specimen section：Antibody I is added dropwise on slide, places into and is incubated in wet box, together with incubation
Wet box is put into 2-8 DEG C of cold storage refrigerator and stands 8-10h together, takes out, and 30min is stood in room temperature, and PBS is rinsed 2-3 times, is added dropwise anti-
Body II, place into and be incubated in wet box, be put into together with incubation wet box in 37.2 DEG C of insulating boxs and stand 40min, taken out, in room temperature
30min is stood, PBS is rinsed 2-3 times；
(7) Pathologic specimen section colour developing：DAB colour developing 3-5min, the intensity of dyeing is controlled under the microscope.
(8) Pathologic specimen section is redyed：Haematoxylin dye liquor redyes 1min, and hydrochloride alcohol differentiation 3s, PBS rinse 5-10 points
Clock.
(9) Pathologic specimen section mounting and microscopy.
Preferably, the antigen retrieval buffers used in the step (4) is EDTA antigen retrieval buffers, volume 1400-
1500ml。
Preferably, PBS is rinsed and is rinsed 5min every time in the step (4).
Preferably, then broken up in the step (5) using haematoxylin with hydrochloride alcohol.
Preferably, PBS is rinsed and is rinsed 3-5min every time in the step (6).
Preferably, colour developing is developed the color using DBA nitrite ions in the step (7), and the DBA nitrite ions colour developing is by DAKO
The μ l of ChemMate EnVision reagent B2ml and DAKOChemMate EnVision reagent Cs 10 are mixed.
Preferably, PBS is rinsed 2-3 times in the step (8).
A kind of Pathologic specimen section SABC operating system, for realizing claim 1-7 any one operating methods,
The system includes：
Section module：Pathologic specimen paraffin is thinly sliced using paraffin slicing machine；
Roasting piece module：Pathologic specimen paraffin section is dried using apparatus for baking；
Dewax module：The paraffin in Pathologic specimen paraffin section is removed using dewaxing cylinder；
Antigen retrieval module：Heated using pressure cooker by the exposure and amendment of antigen；
Staining modules：Pathologic specimen paraffin section is dyed using haematoxylin；
Antibody moiety is added dropwise：Antibody is added dropwise to Pathologic specimen paraffin section using draw a circle pen and droppers of DAKO；
Develop the color module：Pathologic specimen paraffin section is developed the color using dropper；
Mounting microscopy module：For carrying out mounting and microscopy to Pathologic specimen paraffin section.
Preferably, it is additionally provided between the colour developing module and the mounting microscopy module and redyes module.
Compared with prior art, the present invention achieves following beneficial effect：The present invention is used and first dyed with haematoxylin
Antibody I, antibody II is added dropwise, beneficial to the colour developing of antibody, and the size and location that antibody is easy to hold tissue is instilled after dyeing, reduced
The deviation held during antibody to position is added dropwise, is easy to Experimental Standardization to operate, there is stronger actual application value, can effectively keep away
Exempt from actual waste, also can largely reduce the error of experimental data.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, forms the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the system structure diagram of the present invention.
Embodiment
Some vocabulary has such as been used to censure specific components among specification and claim.Those skilled in the art should
It is understood that hardware manufacturer may call same component with different nouns.This specification and claims are not with name
The difference of title is used as the mode for distinguishing component, but is used as the criterion of differentiation with the difference of component functionally.Specification
Subsequent descriptions for implement the application better embodiment, so it is described description be for the purpose of the rule for illustrating the application,
It is not limited to scope of the present application.The protection domain of the application is worked as to be defined depending on appended claims institute defender.
The application is described in further detail below in conjunction with accompanying drawing, but not as the restriction to the application.
Embodiment one：
A kind of Pathologic specimen section SABC operating method, including following steps：
(1) Pathologic specimen paraffin section：Produced Pathologic specimen paraffin is taken, 3 μm of paraffin is cut into paraffin slicing machine
Section；
(2) Pathologic specimen bakes piece：The slide 2h with Pathologic specimen paraffin section is baked in 60 DEG C of insulating boxs, by pathology mark
This paraffin section is dried；
(3) Pathologic specimen section dewaxing：First the slide with Pathologic specimen paraffin section is put at room temperature before dewaxing
60min is put, allows it to cool, recovers, as the same temperature of room temperature, to be soaked with dimethylbenzene I, dimethylbenzene II, dimethylbenzene III afterwards, often
Secondary 5min, then slide is placed in absolute alcohol I, absolute alcohol II, absolute alcohol III and soaked, each 2min, then successively
Move in 95% alcohol I, 95% alcohol II, 85% alcohol and soak, each 2min, finally rinse 2min with PBS, rinse out load glass
Residual solution on piece；
(4) Pathologic specimen section antigen retrieval：By the slide after rinsing well be put into PH in pressure cooker be 8.0 it is anti-
Original repairs 22min in liquid, natural cooling 40min, PBS flushing 2 times, then moves to and 2min is soaked in distilled water, in 3% hydrogen peroxide
8min is soaked, PBS is rinsed 2 times, to block endogenous peroxydase；
(5) Pathologic specimen section statining：Using haematoxylin dyeing 2min；
(6) antibody is added dropwise in Pathologic specimen section：Antibody I is added dropwise on slide, places into and is incubated in wet box, together with incubation
Wet box is put into 6 DEG C of cold storage refrigerators and stands 9h together, takes out, and 30min is stood in room temperature, and PBS is rinsed 2 times, and antibody II is added dropwise, then
It is put into and is incubated in wet box, is put into together with incubation wet box in 37.2 DEG C of insulating boxs and stands 40min, taken out, stood in room temperature
30min, PBS are rinsed 2-3 times；
(7) Pathologic specimen section colour developing：DAB develops the color 4min, controls the intensity of dyeing under the microscope, and endochylema is into brown person
It is judged as positive cell.
(8) Pathologic specimen section redyes haematoxylin dye liquor and redyes 1min, hydrochloride alcohol differentiation 3s, PBS flushing 7 minutes.
(9) Pathologic specimen section mounting and microscopy, microscopy is carried out using microscope.
In the present embodiment, the antigen retrieval buffers used in step (4) is EDTA antigen retrieval buffers, volume 1400-
1500ml。
PBS is rinsed in the present embodiment, in step (4) rinses 5min every time.
In the present embodiment, then broken up in step (5) using haematoxylin with hydrochloride alcohol.
PBS is rinsed in the present embodiment, in step (6) rinses 4min every time.
In the present embodiment, colour developing is using the colour developing of DBA nitrite ions in step (7), and the colour developing of DBA nitrite ions is by DAKO
The μ l of ChemMate EnVision reagent B2ml and DAKOChemMate EnVision reagent Cs 10 are mixed.
In the present embodiment, PBS is rinsed 2 times in step (8).
A kind of Pathologic specimen section SABC operating system, the system include：
Section module：Pathologic specimen paraffin is thinly sliced using paraffin slicing machine；
Roasting piece module：Pathologic specimen paraffin section is dried using apparatus for baking；
Dewax module：The paraffin in Pathologic specimen paraffin section is removed using dewaxing cylinder；
Antigen retrieval module：Heated using pressure cooker by the exposure and amendment of antigen；
Staining modules：Pathologic specimen paraffin section is dyed using haematoxylin；
Antibody moiety is added dropwise：Antibody is added dropwise to Pathologic specimen paraffin section using draw a circle pen and droppers of DAKO；
Develop the color module：Pathologic specimen paraffin section is developed the color using dropper；
Mounting microscopy module：For carrying out mounting and microscopy to Pathologic specimen paraffin section.
In the present embodiment, it is additionally provided between colour developing module and mounting microscopy module and redyes module, redyed using haematoxylin
Dye liquor redyes 1min, hydrochloride alcohol differentiation 3s.
Some preferred embodiments of the application have shown and described in described above, but as previously described, it should be understood that the application
Be not limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and available for various other combinations,
Modification and environment, and above-mentioned teaching or the technology or knowledge of association area can be passed through in application contemplated scope described herein
It is modified., then all should be in this Shen and the change and change that those skilled in the art are carried out do not depart from spirit and scope
Please be in the protection domain of appended claims.
Claims (9)
- The SABC operating method 1. a kind of Pathologic specimen is cut into slices, it is characterised in that including following steps：(1) Pathologic specimen paraffin section：Produced Pathologic specimen paraffin is taken, 2-4 μm of paraffin is cut into paraffin slicing machine and is cut Piece(2) Pathologic specimen bakes piece：The slide 2h with Pathologic specimen paraffin section is baked in 55-65 DEG C of insulating box；(3) Pathologic specimen section dewaxing：First the slide with Pathologic specimen paraffin section is placed at room temperature before dewaxing 60min, soaked afterwards with dimethylbenzene I, dimethylbenzene II, dimethylbenzene III, each 5min, then slide is placed on absolute alcohol I, nothing Soak, each 2min, then moved to successively in 95% alcohol I, 95% alcohol II, 85% alcohol in water-alcohol II, absolute alcohol III Immersion, each 2min, finally rinse 2min with PBS；(4) Pathologic specimen section antigen retrieval：The antigen that the PH that slide after rinsing well is put into pressure cooker is 8.0 is repaiied 20-25min in multiple liquid, more than natural cooling 30min, PBS are rinsed 2-3 times, are then moved to and 2min is soaked in distilled water, 3% pair 8min is soaked in oxygen water, PBS is rinsed 2-3 times；(5) Pathologic specimen section statining：Using haematoxylin dyeing 2-3min；(6) antibody is added dropwise in Pathologic specimen section：Antibody I is added dropwise on slide, places into and is incubated in wet box, together with incubation wet box It is put into together in 2-8 DEG C of cold storage refrigerator and stands 8-10h, take out, 30min is stood in room temperature, PBS is rinsed 2-3 times, and antibody is added dropwise II, place into and be incubated in wet box, be put into together with incubation wet box in 37 DEG C of insulating boxs and stand 40min, taken out, stood in room temperature 30min, PBS are rinsed 2-3 times；(7) Pathologic specimen section colour developing：DAB colour developing 3-5min, the intensity of dyeing is controlled under the microscope.(8) Pathologic specimen section is redyed：Haematoxylin dye liquor redyes 1min, and hydrochloride alcohol differentiation 3s, PBS rinse 5-10 minutes.(9) Pathologic specimen section mounting and microscopy.
- A kind of 2. Pathologic specimen section SABC operating method as claimed in claim 1, it is characterised in that the step (4) antigen retrieval buffers used in is EDTA antigen retrieval buffers, volume 1400-1500ml.
- A kind of 3. Pathologic specimen section SABC operating method as claimed in claim 1, it is characterised in that the step (4) PBS is rinsed in rinses 5min every time.
- A kind of 4. Pathologic specimen section SABC operating method as claimed in claim 1, it is characterised in that the step (5) then broken up in using haematoxylin with hydrochloride alcohol.
- A kind of 5. Pathologic specimen section SABC operating method as claimed in claim 1, it is characterised in that the step (6) PBS is rinsed in rinses 3-5min every time.
- A kind of 6. Pathologic specimen section SABC operating method as claimed in claim 1, it is characterised in that the step (7) colour developing is developed the color using DBA nitrite ions in, and the DBA nitrite ions colour developing is by DAKO ChemMate EnVision reagents B2ml Mixed with the μ l of DAKO ChemMate EnVision reagent Cs 10.
- A kind of 7. Pathologic specimen section SABC operating method as claimed in claim 1, it is characterised in that the step (8) PBS is rinsed 2-3 times in.
- The SABC operating system 8. a kind of Pathologic specimen is cut into slices, for realizing claim 1-7 any one operating methods, its It is characterised by, the system includes：Section module：Pathologic specimen paraffin is thinly sliced using paraffin slicing machine；Roasting piece module：Pathologic specimen paraffin section is dried using apparatus for baking；Dewax module：The paraffin in Pathologic specimen paraffin section is removed using dewaxing cylinder；Antigen retrieval module：Heated using pressure cooker by the exposure and amendment of antigen；Staining modules：Pathologic specimen paraffin section is dyed using haematoxylin；Antibody moiety is added dropwise：Antibody is added dropwise to Pathologic specimen paraffin section using draw a circle pen and droppers of DAKO；Develop the color module：Pathologic specimen paraffin section is developed the color using dropper；Mounting microscopy module：For carrying out mounting and microscopy to Pathologic specimen paraffin section.
- 9. a kind of Pathologic specimen section SABC operating system as claimed in claim 8, it is characterised in that in the colour developing It is additionally provided between module and the mounting microscopy module and redyes module.
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Cited By (5)
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| CN110132695A (en) * | 2019-05-27 | 2019-08-16 | 福州迈新生物技术开发有限公司 | A kind of pathological staining system reduction human error is caused to dye abnormal method |
| CN110411808A (en) * | 2019-08-05 | 2019-11-05 | 李海南 | A kind of quick frozen-section immunohistochemical staining method in art |
| CN110763536A (en) * | 2019-11-01 | 2020-02-07 | 吉林大学 | Lymph node or affected tissue biopsy pathology inspection device |
| CN111766125A (en) * | 2020-07-29 | 2020-10-13 | 广州金域医学检验中心有限公司 | Staining method using fluorescence quenching time difference, automatic staining apparatus, device, and medium |
| CN111912995A (en) * | 2020-08-10 | 2020-11-10 | 英诺维尔智能科技（苏州）有限公司 | High-flux flexible tissue sample analysis system |
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