Application of the easy to operate groupization pen in the detection of cell climbing sheet immunohistochemistry
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
Being 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 when dyeing and spreads, improve the multipurpose immunohistochemistry pen of service speed in cell climbing sheet
Application in Immunohistochemical detection.
Technical solution of the invention is:
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)It is incubated 10 min 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 cleans sample 3 times;
(14)DAB develops the color, and is protected from light, under the microscope;
(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%.
It under external force can be by the bottom of ware body before the abjection of rectangle frame trace line chase, first with recessed along sharp keen cutter
Ditch is drawn, is hooked, and then adds to be deviate from the single hole slot bottom of ware body with external force in ware bottom surface.
The immunohistochemistry pen includes pen container, and spongioid cylinder pen core is arranged in pen container, ink is perfused in pen container,
Wooden pen core is arranged in spongioid cylinder pen core front end;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 the fore shaft of interior pen cap outer surface, pen cap and fixed interior pen cap are integrated out in closing
Pen container mouth is opened, and plays a part of to propose ink brush.
One angle of porous room culture dish is in bevel-faced form, facilitates bearing mark;An angle for cultivating ware lid is in and culture
The bevel-faced form that ware coordinates in the angle on inclined-plane;Cultivate interleave depth >=10mm of ware 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, mark secondary antibody, lining dye(Haematoxylin)Etc. the reality that need not 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 temperature of its processing of cell culture, the time, the standards such as reagent concentration are consistent, keep 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 of piece is tested, and antibody and reagent dosage can be substantially reduced, and is avoided liquid trickling when dyeing and is spread, carries
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 carrying out on the cell climbing sheet of the histotomy that glass is carrier and polystyrene material carrier
Various immunohistochemical staining experiments, can substantially reduce antibody and reagent dosage, avoid liquid trickling when 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 structural schematic diagram of culture dish of the present invention.
Fig. 2 is the structural schematic diagram of immunohistochemistry pen.
Fig. 3 is ware body bottom abjection schematic diagram.
Specific implementation mode
The 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)It is incubated 10 min 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 cleans sample 3 times;
(14)DAB develops the color, and is protected from light, under the microscope;
(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%.
It under external force can be by the bottom of ware body before the abjection of rectangle frame trace line chase, first with recessed along sharp keen cutter
Ditch is drawn, is hooked, and then adds to be deviate from the single hole slot bottom of ware body with external force in ware bottom surface.
The immunohistochemistry pen includes pen container 5, and spongioid cylinder pen core 6 is arranged in pen container, oil is perfused in pen container
Wooden pen core 10 is arranged in ink, spongioid cylinder pen core front end;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 9 inner lip of outer pen cap, and cylinder pen core end side is equipped with ink brush 11;Outer pen cap
To be equipped with fore shaft inner lip week convex, coincide with recessed match of the fore shaft of interior pen cap outer surface, play pen cap and fixed interior pen cap in closing
It is integrated and opens pen container mouth, and play a part of to propose ink brush.Ink is set on interior pen cap and exchanges hole 12.
One angle of porous room culture dish is in bevel-faced form, facilitates bearing mark;An angle for cultivating ware lid is in and culture
The bevel-faced form that ware coordinates in the angle on inclined-plane;Cultivate interleave depth >=10mm of ware lid and culture dish previous anastomotic periphery.