CN106092702B - The group pen for reducing reagent dosage is applied in the detection of cell climbing sheet immunohistochemistry - Google Patents
The group pen for reducing reagent dosage is applied in the detection of cell climbing sheet immunohistochemistry Download PDFInfo
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- CN106092702B CN106092702B CN201610404342.9A CN201610404342A CN106092702B CN 106092702 B CN106092702 B CN 106092702B CN 201610404342 A CN201610404342 A CN 201610404342A CN 106092702 B CN106092702 B CN 106092702B
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- 230000009194 climbing Effects 0.000 title claims abstract description 26
- 238000003364 immunohistochemistry Methods 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 title claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 8
- 239000011521 glass Substances 0.000 claims abstract description 21
- 238000004043 dyeing Methods 0.000 claims abstract description 14
- 230000001744 histochemical effect Effects 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000004113 cell culture Methods 0.000 claims abstract description 5
- QGJOPFRUJISHPQ-UHFFFAOYSA-N Carbon disulfide Chemical compound S=C=S QGJOPFRUJISHPQ-UHFFFAOYSA-N 0.000 claims description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 10
- 239000012188 paraffin wax Substances 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 238000013115 immunohistochemical detection Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims description 6
- RSWGJHLUYNHPMX-UHFFFAOYSA-N Abietic-Saeure Natural products C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 claims description 5
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 claims description 5
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 claims description 5
- 235000013871 bee wax Nutrition 0.000 claims description 5
- 239000012166 beeswax Substances 0.000 claims description 5
- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 claims description 5
- 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 description 4
- 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 description 4
- 239000004793 Polystyrene Substances 0.000 claims description 4
- 229920002223 polystyrene Polymers 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000001293 FEMA 3089 Substances 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 239000012122 aqueous mounting media Substances 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 239000003350 kerosene Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012224 working solution Substances 0.000 claims description 3
- 239000011435 rock Substances 0.000 claims 1
- 238000011532 immunohistochemical staining Methods 0.000 abstract description 7
- 238000011081 inoculation Methods 0.000 abstract description 2
- 239000011148 porous material Substances 0.000 abstract description 2
- 238000009792 diffusion process Methods 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 235000021355 Stearic acid Nutrition 0.000 description 2
- RWGFKTVRMDUZSP-UHFFFAOYSA-N cumene Chemical group CC(C)C1=CC=CC=C1 RWGFKTVRMDUZSP-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 238000003365 immunocytochemistry Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000008117 stearic acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance 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
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
-
- 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
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- Immunology (AREA)
- Engineering & Computer Science (AREA)
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- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
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- Physics & Mathematics (AREA)
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- Urology & Nephrology (AREA)
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- Microbiology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a kind of application of groupization pen for reducing antibody dosage in the detection of cell climbing sheet immunohistochemistry, and cell inoculation is carried out cell climbing sheet in glass slide formula culture dish, immunocyte histochemical stain identification is carried out after cell culture；The glass slide formula culture dish is equipped with culture dish body and ware lid, and culture dish build number is divided to two kinds of single hole room and porous room；Each pore chamber area is more than glass slide, and ware body bottom is used directly as glass slide, and ware body bottom lateral surface is equipped with rectangle frame trace line chase corresponding with glass slide size, under external force can deviate from the bottom of ware body along rectangle frame trace line chase.The various immunohistochemical stainings that the present invention is suitable for glass slice are tested, and can substantially reduce antibody and reagent dosage, and liquid trickling and diffusion, improve service speed when avoiding dyeing.It is especially applicable for carrying out extensive, the multigroup other immunohistochemical staining of large sample in the experimental study of cell climbing sheet or cell smear.
Description
The application is application number：201410500701.1, the applying date：2014.9.26, title：" multipurpose immunohistochemistry pen
The divisional application of application in cell climbing sheet Immunohistochemical detection ".
Technical field
The present invention relates to a kind of application of immunohistochemistry pen in cell climbing sheet Immunohistochemical detection.
Background technology
With specific antibody to the labels of certain chemical constituents analysis in histotomy and its cell climbing sheet and its content into
Row tissue and cell in-situ are qualitative, position or quantitative study, this technology are known as immunocytochemistry
（immunocytochemistry）Technology.
Usual cell climbing sheet Immunohistochemical detection is to carry out dying operation one by one to individual cell climbing sheet, generally
A kind of antibody, the detection of albumen and gene can only be done to single sample, it is difficult to which competent extensive, multisample and multigroup other medicine are real
Test scientific research.
Invention content
It is suitable for various immunohistochemical stainings the purpose of the present invention is to provide one kind to test, antibody can be substantially reduced
And reagent dosage, it avoids liquid trickling during dyeing and spreads, improve the multipurpose immunohistochemistry pen of service speed in cell climbing sheet
Application in Immunohistochemical detection.
The present invention technical solution be：
A kind of application of multipurpose immunohistochemistry pen in cell climbing sheet Immunohistochemical detection, it is characterized in that：It will be thin
Born of the same parents are inoculated in glass slide formula culture dish and carry out cell climbing sheet, and immunocyte histochemical stain mirror is carried out after cell culture
It is fixed；The glass slide formula culture dish is equipped with culture dish body and ware lid, and culture dish build number is divided to two kinds of single hole room and porous room；Each hole
Room area is more than glass slide, and ware body bottom is used directly as glass slide, and ware body bottom lateral surface is equipped with corresponding with glass slide size
Rectangle frame trace line chase under external force can deviate from the bottom of ware body along rectangle frame trace line chase；
The immunocyte histochemical stain identification includes the following steps successively：
（1）15 min are fixed with ice acetone or 4% paraformaldehyde is fixed；
（2）Flowing water rinses, and sample is cleaned 3 times with PBS；
（3）10 min are incubated with 0.5%Triton X-100；
（4）0.3%H2O2It is incubated 10 min；
（5）After cleaning sample 3 times with PBS, hot wind is fully dry；
（7）Being drawn ink on cell climbing sheet with immunohistochemistry pen divides 2-20 to separate dyeing area；
（8）Air is fully dry；
（9）It is closed with normal two antiserum and is incubated 10 min；
（10）Mouse is added dropwise respectively in each cell dyeing area or rabbit-anti first antibody is incubated 30~60 min；
（11）It absorbs each cell dyeing area liquid and cleans sample 3 times with PBS；
（12）Enzyme-linked mouse is added dropwise or rabbit secondary antibody working solution is incubated 30~60 min；
（13）PBS cleanings sample 3 times；
（14）DAB develops the color, and is protected from light, Microscopic observation；
（15）Distillation washing；
（16）Haematoxylin lining dye；
（17）Hydrochloride alcohol breaks up, and originally washes；
（18）Aqueous mounting medium mounting.
The ink is prepared from the following ingredients in percentage by mixing composition：Rosin 2 ~ 6%, beeswax 18 ~ 26%, paraffin 3 ~ 8%,
Isopropyl ether 5 ~ 10%, dichloromethane 5 ~ 10%, gasoline 25 ~ 30%, carbon disulfide 12 ~ 17%, carbon tetrachloride 2 ~ 6%, chloroform 2 ~ 6%, ring
Ketone 2 ~ 6%；The sum of above-mentioned each component dosage is 100%；
Or the ink is made of following component mixing：Polystyrene 2 ~ 4%, DMF9 ~ 11%, THF9 ~ 11%, rosin 2 ~ 4%,
Beeswax 13 ~ 16%, paraffin 2 ~ 4%, isopropyl ether 4 ~ 6%, dichloromethane 4 ~ 6%, gasoline 18 ~ 22%, carbon disulfide 9 ~ 11%, carbon tetrachloride
9 ~ 11%, chloroform 1 ~ 2%, cyclohexanone 1 ~ 2%, kerosene 2 ~ 4%, turpentine oil 1 ~ 2%；The sum of above-mentioned each component dosage is 100%；
Or the ink is made of following component mixing：Stearic acid 30 ~ 36%, 15 ~ 20%, 46 DEG C of paraffin 9 ~ 15% of benzene, isopropyl
Ether 7 ~ 11%, carbon disulfide 15 ~ 20%, chloroform 15 ~ 20%；The sum of above-mentioned each component dosage is 100%.
Before the bottom of ware body can be deviate from along rectangle frame trace line chase under external force, first with recessed along sharp keen cutter
Ditch is drawn, is hooked, and then adds in ware bottom surface and can be deviate from the single hole slot bottom of ware body with external force.
The immunohistochemistry pen includes pen container, and spongioid cylinder pen core is set in pen container, ink is perfused in pen container,
Spongioid cylinder pen core front end sets wooden pen core；Spongioid cylinder pen core is placed in pen container, and pen container is rectangular
Body or cylinder, it is identical that pen container mouth outside has screw thread to be matched with the internal thread of interior pen cap；Stainless shot one is placed in pen container, is shaken
Make the effect for mixing ink when shaking pen container；The interior pen cap rise closing pen container mouth, prevent ink volatilization keep its liquid phase state and
The effect of the outer pen cap of connection, inner surface have the internal thread closed with pen container tone, can spiral cover pen container mouth, the outer surface of interior pen cap is set
There is the fore shaft for matching and coincideing with outer pen cap inner lip recessed, cylinder pen core end side is equipped with ink brush；The inner lip of outer pen cap
It is convex to be equipped with fore shaft week, coincide with recessed match of interior pen cap fore shaft, rise in closing pen cap and it is fixed in pen cap be integrated unlatching pen container mouth,
And play a part of to propose ink brush.
One angle of porous room culture dish is in bevel-faced form, facilitates bearing mark；One angle of culture dish lid is in and culture
The bevel-faced form that ware coordinates in the angle on inclined-plane；Interleave depth >=10mm of culture dish lid and culture dish previous anastomotic periphery.
The present invention is conducive to the dyeing of immunohistochemistry, and staining procedure is similar to Immunohistochemistry, but sample process
Temperature, the time, the standards such as reagent concentration are consistent, and the primary antibody type of label is more, and comparativity and reliability are significantly increased！Processing
Sample size it is more, efficient quick.PBS cleanings, serum are incubated, label secondary antibody, lining dye（Haematoxylin）Etc. the reality for not needing to separate
It tests operation and can synchronize and handled, convenient and efficient, experiment condition standard is consistent and easy to control.It is carried out using large area cell climbing sheet
The standards such as the temperature of its processing of cell culture, time, reagent concentration are consistent, make the comparativity of experimental result and reliability aobvious
It writes and improves.The quantity of Tissue Culture Dish hole slot is reduced, easy to operate, efficient quick.Specific ink formulations have fully ensured that work
Make effect.It is tested suitable for various immunohistochemical stainings, including；Paraffin tissue sections, frozen tissue section and cell are climbed
The immunohistochemical staining experiment of piece, can substantially reduce antibody and reagent dosage, avoid liquid trickling during dyeing and spread, carry
High service speed；It is especially applicable for carrying out in the experimental study of cell climbing sheet or cell smear on a large scale, large sample is multigroup not
Immunohistochemical staining.
The group pen is suitable for glass to be carried out on the histotomy of carrier and the cell climbing sheet of polystyrene material carrier
Various immunohistochemical staining experiments, can substantially reduce antibody and reagent dosage, avoid liquid trickling during dyeing and spread, carry
High service speed.It can carry out extensive, multisample and multigroup other medical experiment scientific research.
Description of the drawings
The invention will be further described with reference to the accompanying drawings and examples.
Fig. 1 is the structure diagram of culture dish of the present invention.
Fig. 2 is the structure diagram of immunohistochemistry pen.
Fig. 3 is ware body bottom abjection schematic diagram.
Specific embodiment
Application of a kind of multipurpose immunohistochemistry pen in cell climbing sheet Immunohistochemical detection, by cell inoculation in load
Cell climbing sheet is carried out in slide formula culture dish, immunocyte histochemical stain identification is carried out after cell culture；The load
Slide formula culture dish is equipped with culture dish body 1 and ware lid 2, and culture dish build number is divided to two kinds of single hole room and porous room；Each pore chamber area
More than glass slide, ware body bottom 3 is used directly as glass slide, and ware body bottom lateral surface is equipped with rectangle corresponding with glass slide size
Frame trace line chase 4 under external force can deviate from the bottom of ware body along rectangle frame trace line chase；
The immunocyte histochemical stain identification includes the following steps successively：
（1）15 min are fixed with ice acetone or 4% paraformaldehyde is fixed；
（2）Flowing water rinses, and sample is cleaned 3 times with PBS；
（3）10 min are incubated with 0.5%Triton X-100；
（4）0.3%H2O2It is incubated 10 min；
（5）After cleaning sample 3 times with PBS, hot wind is fully dry；
（7）Being drawn ink on cell climbing sheet with immunohistochemistry pen divides 2-20 to separate dyeing area；
（8）Air is fully dry；
（9）It is closed with normal two antiserum and is incubated 10 min；
（10）Mouse is added dropwise respectively in each cell dyeing area or rabbit-anti first antibody is incubated 30~60 min；
（11）It absorbs each cell dyeing area liquid and cleans sample 3 times with PBS；
（12）Enzyme-linked mouse is added dropwise or rabbit secondary antibody working solution is incubated 30~60 min；
（13）PBS cleanings sample 3 times；
（14）DAB develops the color, and is protected from light, Microscopic observation；
（15）Distillation washing；
（16）Haematoxylin lining dye；
（17）Hydrochloride alcohol breaks up, and originally washes；
（18）Aqueous mounting medium mounting.
The ink is prepared from the following ingredients in percentage by mixing composition：Rosin 2 ~ 6%, beeswax 18 ~ 26%, paraffin 3 ~ 8%,
Isopropyl ether 5 ~ 10%, dichloromethane 5 ~ 10%, gasoline 25 ~ 30%, carbon disulfide 12 ~ 17%, carbon tetrachloride 2 ~ 6%, chloroform 2 ~ 6%, ring
Ketone 2 ~ 6%；The sum of above-mentioned each component dosage is 100%；
Or the ink is made of following component mixing：Polystyrene 2 ~ 4%, DMF9 ~ 11%, THF9 ~ 11%, rosin 2 ~ 4%,
Beeswax 13 ~ 16%, paraffin 2 ~ 4%, isopropyl ether 4 ~ 6%, dichloromethane 4 ~ 6%, gasoline 18 ~ 22%, carbon disulfide 9 ~ 11%, carbon tetrachloride
9 ~ 11%, chloroform 1 ~ 2%, cyclohexanone 1 ~ 2%, kerosene 2 ~ 4%, turpentine oil 1 ~ 2%；The sum of above-mentioned each component dosage is 100%；
Or the ink is made of following component mixing：Stearic acid 30 ~ 36%, 15 ~ 20%, 46 DEG C of paraffin 9 ~ 15% of benzene, isopropyl
Ether 7 ~ 11%, carbon disulfide 15 ~ 20%, chloroform 15 ~ 20%；The sum of above-mentioned each component dosage is 100%.
Before the bottom of ware body can be deviate from along rectangle frame trace line chase under external force, first with recessed along sharp keen cutter
Ditch is drawn, is hooked, and then adds in ware bottom surface and can be deviate from the single hole slot bottom of ware body with external force.
The immunohistochemistry pen includes pen container 5, and spongioid cylinder pen core 6 is set in pen container, oil is perfused in pen container
Ink, spongioid cylinder pen core front end set wooden pen core 10；Spongioid cylinder pen core is placed in pen container, and pen container is
Cuboid or cylinder, it is identical that pen container mouth outside has screw thread to be matched with the internal thread of interior pen cap 7；Stainless shot 8 is placed in pen container
One, make the effect for mixing ink when rocking pen container；The interior pen cap plays closing pen container mouth, prevents ink volatilization from keeping its liquid phase
State and the effect for coupling outer pen cap, inner surface have the internal thread closed with pen container tone, can spiral cover pen container mouth, interior pen cap outside
Surface is recessed equipped with identical fore shaft is matched with outer 9 inner lip of pen cap, and cylinder pen core end side is equipped with ink brush 11；Outer pen cap
Inner lip week be equipped with fore shaft it is convex, with interior pen cap fore shaft it is recessed match coincide, rise closing in pen cap and fix in pen cap be integrated out
Pen container mouth is opened, and plays a part of to propose ink brush.Ink is set to exchange hole 12 on interior pen cap.
One angle of porous room culture dish is in bevel-faced form, facilitates bearing mark；One angle of culture dish lid is in and culture
The bevel-faced form that ware coordinates in the angle on inclined-plane；Interleave depth >=10mm of culture dish lid and culture dish previous anastomotic periphery.
Claims (4)
1. a kind of application of groupization pen for reducing antibody dosage in the detection of cell climbing sheet immunohistochemistry, it is characterized in that：By cell
It is inoculated in glass slide formula culture dish and carries out cell climbing sheet, immunocyte histochemical stain mirror is carried out after cell culture
It is fixed；The glass slide formula culture dish is equipped with culture dish body and ware lid, and culture dish build number is divided to two kinds of single hole room and porous room；Each hole
Room area is more than glass slide, and ware body bottom is used directly as glass slide, and ware body bottom lateral surface is equipped with corresponding with glass slide size
Rectangle frame trace line chase under external force can deviate from the bottom of ware body along rectangle frame trace line chase, and acquisition has culture
Cell face is ready for use on various Immunohistochemical detections just as the ware bottom of glass slide；
The immunocyte histochemical stain identification includes the following steps successively：
（1）15 min are fixed with ice acetone or 4% paraformaldehyde is fixed；
（2）Flowing water rinses, and sample is cleaned 3 times with PBS；
（3）10 min are incubated with 0.5%Triton X-100；
（4）0.3%H2O2It is incubated 10 min；
（5）After cleaning sample 3 times with PBS, hot wind is fully dry；
（7）Being drawn ink on cell climbing sheet with immunohistochemistry pen divides 2-20 to separate dyeing area；
（8）Air is fully dry；
（9）It is closed with normal two antiserum and is incubated 10 min；
（10）Mouse is added dropwise respectively in each cell dyeing area or rabbit-anti first antibody is incubated 30~60 min；
（11）It absorbs each cell dyeing area liquid and cleans sample 3 times with PBS；
（12）Enzyme-linked mouse is added dropwise or rabbit secondary antibody working solution is incubated 30~60 min；
（13）PBS cleanings sample 3 times；
（14）DAB develops the color, and is protected from light, Microscopic observation；
（15）Distillation washing；
（16）Haematoxylin lining dye；
（17）Hydrochloride alcohol breaks up, and originally washes；
（18）Aqueous mounting medium mounting；
The ink is made of following component mixing：Polystyrene 2 ~ 4%, DMF9 ~ 11%, THF9 ~ 11%, rosin 2 ~ 4%, beeswax 13
~ 16%, paraffin 2 ~ 4%, isopropyl ether 4 ~ 6%, dichloromethane 4 ~ 6%, gasoline 18 ~ 22%, carbon disulfide 9 ~ 11%, carbon tetrachloride 9 ~ 11%,
Chloroform 1 ~ 2%, cyclohexanone 1 ~ 2%, kerosene 2 ~ 4%, turpentine oil 1 ~ 2%；The sum of above-mentioned each component dosage is 100%；
The immunohistochemistry pen includes pen container, and spongioid cylinder pen core is set in pen container, ink, sponge are perfused in pen container
The cylinder pen core front end of sample sets wooden pen core；Spongioid cylinder pen core is placed in pen container, pen container for cuboid or
Cylinder, it is identical that pen container mouth outside has screw thread to be matched with the internal thread of interior pen cap；Stainless shot one is placed in pen container, rocks pen
Make the effect for mixing ink during cylinder；The interior pen cap plays closing pen container mouth, prevents ink volatilization from keeping its liquid phase state and connection
The effect of outer pen cap, inner surface have the internal thread closed with pen container tone, can spiral cover pen container mouth, the outer surface of interior pen cap be equipped with
The identical fore shaft of outer pen cap inner lip matching is recessed, and cylinder pen core end side is equipped with ink brush；The inner lip week of outer pen cap sets
Have that fore shaft is convex, coincide with recessed match of the fore shaft of interior pen cap, rise in closing pen cap and it is fixed in pen cap be integrated and open pen container mouth, and
Play a part of to propose ink brush；
Before the bottom of ware body can be deviate from along rectangle frame trace line chase under external force, first with chase along sharp keen cutter
It draws, hook, then the single hole slot bottom of ware body can slightly be deviate from external force in ware bottom surface.
2. application of the groupization pen according to claim 1 for reducing antibody dosage in the detection of cell climbing sheet immunohistochemistry,
It is characterized in that：One angle of porous room culture dish is in bevel-faced form, facilitates bearing mark.
3. application of the groupization pen according to claim 2 for reducing antibody dosage in the detection of cell climbing sheet immunohistochemistry,
It is characterized in that：One angle of culture dish lid is in the bevel-faced form coordinated with culture dish in the angle on inclined-plane.
4. application of the groupization pen according to claim 3 for reducing antibody dosage in the detection of cell climbing sheet immunohistochemistry,
It is characterized in that：Interleave depth >=10mm of culture dish lid and culture dish previous anastomotic periphery.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610404342.9A CN106092702B (en) | 2014-09-26 | 2014-09-26 | The group pen for reducing reagent dosage is applied in the detection of cell climbing sheet immunohistochemistry |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610404342.9A CN106092702B (en) | 2014-09-26 | 2014-09-26 | The group pen for reducing reagent dosage is applied in the detection of cell climbing sheet immunohistochemistry |
| CN201410500701.1A CN104359741B (en) | 2014-09-26 | 2014-09-26 | Application of multi-functional PAP pen in cell climbing immunohistochemical detection |
Related Parent Applications (1)
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| CN105865879A (en) * | 2016-05-19 | 2016-08-17 | 四川金域医学检验中心有限公司 | Operation method for immunohistochemical staining of frozen sections |
| CN106248464B (en) * | 2016-09-02 | 2020-06-09 | 四川大学 | Cell climbing sheet efficient dyeing device |
| CN107699486A (en) * | 2017-09-22 | 2018-02-16 | 山东省农业科学院畜牧兽医研究所 | For dyeing the Tissue Culture Dish and cell culture dyeing, observational technique of observation |
| CN109100503B (en) * | 2018-09-20 | 2021-02-02 | 同济大学 | Immunohistochemical pen liquid and preparation method thereof |
| CN110887720A (en) * | 2019-12-11 | 2020-03-17 | 武汉原谷生物科技有限责任公司 | Immunohistochemical pen semisolid pen paste and preparation method thereof |
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| Publication number | Publication date |
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| CN106092703B (en) | 2018-07-17 |
| CN104359741A (en) | 2015-02-18 |
| CN106092702A (en) | 2016-11-09 |
| CN105938064B (en) | 2019-03-19 |
| CN106092703A (en) | 2016-11-09 |
| CN105938065B (en) | 2019-03-12 |
| CN105938064A (en) | 2016-09-14 |
| CN104359741B (en) | 2017-01-11 |
| CN105938065A (en) | 2016-09-14 |
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