Summary of the invention
It is an object of the invention to provide one and be applicable to the experiment of various immunohistochemical staining, antibody and reagent dosage can be substantially reduced, liquid trickling and diffusion when avoiding dyeing, the application in cell climbing sheet Immunohistochemical detection of the multipurpose SABC pen of raising speed of operation.
The technical solution of the present invention is:
The application in cell climbing sheet Immunohistochemical detection of a kind of multipurpose SABC pen, is characterized in that: is inoculated in by cell in microscope slide formula culture dish and carries out cell climbing sheet, and cell is cultivated after terminating and carried out immunocyte histochemical stain qualification;Described microscope slide formula culture dish is provided with culture dish body and ware lid, culture dish build number point single hole room and many pore chambers two kinds;Each pore chamber area is more than microscope slide, uses directly as microscope slide at the bottom of ware body, and lateral surface at the bottom of ware body is provided with the rectangle frame trace line chase corresponding with microscope slide size, can be deviate from along rectangle frame trace line chase the bottom of ware body under external force;
Described immunocyte histochemical stain is identified and is comprised the following steps: successively
(1) fix 15 min with ice acetone or 4% paraformaldehyde is fixed;
(2) flowing water rinsing, by PBS specimen 3 times;
(3) 10 min are hatched with 0.5%Triton X-100;
(4) 0.3%H2O2Hatch 10 min;
(5) with after PBS specimen 3 times, hot blast is the driest;
(7) with SABC pen, ink is drawn on cell climbing sheet a point 2-20 separation dyeing district;
(8) air is the driest;
(9) 10 min are hatched with normal two antiserums closings;
(10) drip mice respectively in each cell dyeing district or the anti-first antibody of rabbit hatches 30~60 min;
(11) each cell dyeing district liquid is absorbed and by PBS specimen 3 times;
(12) dropping enzyme connection mice or rabbit second antibody working solution hatch 30~60 min;
(13) PBS specimen 3 times;
(14) DAB colour developing, lucifuge, Microscopic observation;
(15) distillation washing;
(16) haematoxylin lining dye;
(17) hydrochloride alcohol differentiation, washes from the beginning;
(18) aqueous mounting medium mounting.
Described ink is prepared from the following ingredients in percentage by mixing and forms: Colophonium 2 ~ 6%, Cera Flava 18 ~ 26%, paraffin 3 ~ 8%, diisopropyl ether 5 ~ 10%, dichloromethane 5 ~ 10%, gasoline 25 ~ 30%, Carbon bisulfide 12 ~ 17%, carbon tetrachloride 2 ~ 6%, chloroform 2 ~ 6%, cyclohexanone 2 ~ 6%;Above-mentioned each amounts of components sum is 100%;
Or described ink is made up of following component mixing: polystyrene 2 ~ 4%, DMF9 ~ 11%, THF9 ~ 11%, Colophonium 2 ~ 4%, Cera Flava 13 ~ 16%, paraffin 2 ~ 4%, diisopropyl ether 4 ~ 6%, dichloromethane 4 ~ 6%, gasoline 18 ~ 22%, Carbon bisulfide 9 ~ 11%, carbon tetrachloride 9 ~ 11%, chloroform 1 ~ 2%, cyclohexanone 1 ~ 2%, kerosene 2 ~ 4%, Oleum Terebinthinae 1 ~ 2%;Above-mentioned each amounts of components sum is 100%;
Or described ink is made up of following component mixing: stearic acid 30 ~ 36%, benzene 15 ~ 20%, 46 DEG C of paraffin 9 ~ 15%, diisopropyl ether 7 ~ 11%, Carbon bisulfide 15 ~ 20%, chloroform 15 ~ 20%;Above-mentioned each amounts of components sum is 100%.
Before the bottom of ware body can being deviate from along rectangle frame trace line chase under external force, first draw with sharp keen cutter chase along the line, hook, then add in ware bottom surface and the single hole bottom land of ware body can be deviate from by external force.
Described SABC pen includes pen container, arranges spongioid cylinder pen core in pen container, is perfused with ink in pen container, and spongioid cylinder pen core front end arranges wooden pen core;Spongioid cylinder pen core is placed in pen container, and pen container is cuboid or cylinder, has screw thread to mate with the female thread of the interior cap for brush and coincide outside pen container mouth;Pen container is placed stainless shot one, when rocking pen container, makees to mix the effect of ink;The described interior cap for brush plays closing pen container mouth, prevent ink volatilization from keeping its liquid phase state and coupling the effect of the outer cap for brush, its inner surface has the female thread closed with pen container tone, can spiral cover pen container mouth, the outer surface of the interior cap for brush be provided with mate with outer cap for brush inner lip coincide fore shaft recessed, cylinder pen core end side is provided with ink brush;It is recessed that the inner lip week of the outer cap for brush is provided with fore shaft, mates with interior cap for brush outer lip and coincide, and in a closing, the cap for brush and the fixing interior cap for brush are integrated and open pen container mouth, and act the effect proposing ink brush.
One angle of many pore chambers culture dish is bevel-faced form, facilitates bearing mark;One angle of culture dish lid is in the bevel-faced form coordinated with the angle that culture dish is inclined-plane;Culture dish lid and the interleave depth >=10mm of culture dish anastomotic stoma periphery.
The standards such as the present invention is conducive to the dyeing of SABC, and staining procedure is similar to Immunohistochemistry, but the temperature of sample process, the time, reagent concentration are consistent, and an anti-kind of labelling is many, and comparability and reliability are significantly increased!The sample size processed is many, efficient quick.PBS, sera incubation, labelling two resist, and the experimental implementation that lining dye (haematoxylin) etc. need not separate can synchronize to process, and convenient and swift, experiment condition standard is consistent and easy to control.Using large area cell climbing sheet to carry out cell and cultivate its temperature processed, time, the standard such as reagent concentration is consistent, makes the comparability of experimental result and reliability be significantly increased.The quantity of Tissue Culture Dish hole slot reduces, easy to operate, efficient quick.Specific ink formulations has fully ensured that working effect.It is applicable to the experiment of various immunohistochemical staining, including;The immunohistochemical staining experiment of paraffin tissue sections, frozen tissue section and cell climbing sheet, can substantially reduce antibody and reagent dosage, it is to avoid liquid trickling and diffusion during dyeing, improves speed of operation;Be especially applicable in the experimentation of cell climbing sheet or cell smear carrying out extensive, the many groups of large sample immunohistochemical staining.
This group pen is applicable on the cell climbing sheet of tissue slice that glass is carrier and polystyrene material carrier carry out various immunohistochemical staining experiment, can substantially reduce antibody and reagent dosage, it is to avoid liquid trickling and diffusion during dyeing, improves speed of operation.Can carry out extensive, the medical experiment scientific research of multisample and many groups.
Detailed description of the invention
The application in cell climbing sheet Immunohistochemical detection of a kind of multipurpose SABC pen, is inoculated in cell in microscope slide formula culture dish and carries out cell climbing sheet, and cell is cultivated after terminating and carried out immunocyte histochemical stain qualification;Described microscope slide formula culture dish is provided with culture dish body 1 and ware lid 2, culture dish build number point single hole room and many pore chambers two kinds;Each pore chamber area is more than microscope slide, and at the bottom of ware body, 3 use directly as microscope slide, and lateral surface at the bottom of ware body is provided with the rectangle frame trace line chase 4 corresponding with microscope slide size, can be deviate from along rectangle frame trace line chase the bottom of ware body under external force;
Described immunocyte histochemical stain is identified and is comprised the following steps: successively
(1) fix 15 min with ice acetone or 4% paraformaldehyde is fixed;
(2) flowing water rinsing, by PBS specimen 3 times;
(3) 10 min are hatched with 0.5%Triton X-100;
(4) 0.3%H2O2Hatch 10 min;
(5) with after PBS specimen 3 times, hot blast is the driest;
(7) with SABC pen, ink is drawn on cell climbing sheet a point 2-20 separation dyeing district;
(8) air is the driest;
(9) 10 min are hatched with normal two antiserums closings;
(10) drip mice respectively in each cell dyeing district or the anti-first antibody of rabbit hatches 30~60 min;
(11) each cell dyeing district liquid is absorbed and by PBS specimen 3 times;
(12) dropping enzyme connection mice or rabbit second antibody working solution hatch 30~60 min;
(13) PBS specimen 3 times;
(14) DAB colour developing, lucifuge, Microscopic observation;
(15) distillation washing;
(16) haematoxylin lining dye;
(17) hydrochloride alcohol differentiation, washes from the beginning;
(18) aqueous mounting medium mounting.
Described ink is prepared from the following ingredients in percentage by mixing and forms: Colophonium 2 ~ 6%, Cera Flava 18 ~ 26%, paraffin 3 ~ 8%, diisopropyl ether 5 ~ 10%, dichloromethane 5 ~ 10%, gasoline 25 ~ 30%, Carbon bisulfide 12 ~ 17%, carbon tetrachloride 2 ~ 6%, chloroform 2 ~ 6%, cyclohexanone 2 ~ 6%;Above-mentioned each amounts of components sum is 100%;
Or described ink is made up of following component mixing: polystyrene 2 ~ 4%, DMF9 ~ 11%, THF9 ~ 11%, Colophonium 2 ~ 4%, Cera Flava 13 ~ 16%, paraffin 2 ~ 4%, diisopropyl ether 4 ~ 6%, dichloromethane 4 ~ 6%, gasoline 18 ~ 22%, Carbon bisulfide 9 ~ 11%, carbon tetrachloride 9 ~ 11%, chloroform 1 ~ 2%, cyclohexanone 1 ~ 2%, kerosene 2 ~ 4%, Oleum Terebinthinae 1 ~ 2%;Above-mentioned each amounts of components sum is 100%;
Or described ink is made up of following component mixing: stearic acid 30 ~ 36%, benzene 15 ~ 20%, 46 DEG C of paraffin 9 ~ 15%, diisopropyl ether 7 ~ 11%, Carbon bisulfide 15 ~ 20%, chloroform 15 ~ 20%;Above-mentioned each amounts of components sum is 100%.
Before the bottom of ware body can being deviate from along rectangle frame trace line chase under external force, first draw with sharp keen cutter chase along the line, hook, then add in ware bottom surface and the single hole bottom land of ware body can be deviate from by external force.
Described SABC pen includes pen container 5, arranges spongioid cylinder pen core 6 in pen container, is perfused with ink in pen container, and spongioid cylinder pen core front end arranges wooden pen core 10;Spongioid cylinder pen core is placed in pen container, and pen container is cuboid or cylinder, has screw thread to mate with the female thread of the interior cap for brush 7 and coincide outside pen container mouth;Pen container is placed stainless shot 8 one, when rocking pen container, makees to mix the effect of ink;The described interior cap for brush plays closing pen container mouth, prevent ink volatilization from keeping its liquid phase state and coupling the effect of the outer cap for brush, its inner surface has the female thread closed with pen container tone, can spiral cover pen container mouth, the outer surface of the interior cap for brush be provided with mate with the outer cap for brush 9 inner lip coincide fore shaft recessed, cylinder pen core end side is provided with ink brush 11;It is recessed that the inner lip week of the outer cap for brush is provided with fore shaft, mates with interior cap for brush outer lip and coincide, and in a closing, the cap for brush and the fixing interior cap for brush are integrated and open pen container mouth, and act the effect proposing ink brush.Ink exchange hole 12 is set on the interior cap for brush.
One angle of many pore chambers culture dish is bevel-faced form, facilitates bearing mark;One angle of culture dish lid is in the bevel-faced form coordinated with the angle that culture dish is inclined-plane;Culture dish lid and the interleave depth >=10mm of culture dish anastomotic stoma periphery.