CN213800816U - Immunocytochemistry four-staining detection kit - Google Patents

Abstract

The utility model discloses an immunocytochemistry four dyes method detect reagent box, including box body 1, the inside of box body 1 is provided with liner 2, and container hole 3 has been seted up at the top of liner 2, and the quantity of container hole 3 is seven, and aseptic device hole 4 that is located container hole 3 right side has been seted up at the top of liner 2, and the quantity in aseptic device hole 4 is four. The utility model discloses compact structure, convenient to carry, good reproducibility, labour saving and time saving, P16 Ki-67, MCM2 TOP 2A's four staining of immunocytochemistry detect the high efficiency, and is stable, can be fine satisfy the science, the needs of work, the method of having solved the single through immunocytochemistry only detects P16, Ki-67 gene or only detect MCM2, the method of TOP2A gene, it wastes time and energy to detect, need two samples of simultaneous processing, then detect the acquisition result, it is comparatively complicated, the dyeing liquid is easy old, and the dyeing effect is unstable, influence the accuracy that the piece was read to laboratory staff's pathology, be not convenient for observe, the problem that the accuracy of detection is low.

Description

Immunocytochemistry four-staining detection kit

Technical Field

The utility model relates to a kit technical field specifically is immunocytochemistry four-stain method detect reagent box.

Background

The p16 gene is also named as a multi-tumor cancer suppressor gene, the expressed cancer suppressor protein p16 is a negative regulatory protein of cell cycle progression, when the cervical epithelial cells are integrally infected by the human papilloma virus, the oncogene E7 is introduced and activated, the oncogenic protein E7 is promoted to be expressed, pRB is inhibited to be combined with E2F protein, and the proliferation of the cervical epithelial cells is promoted; when the cell is over-proliferated, the negative feedback regulation mechanism of cell proliferation is activated, namely the tumor suppressor gene p16 is activated to promote the expression of tumor suppressor protein p16 and promote the combination of pRB and E2F protein.

In cervical cancer screening, a method of only detecting P16 and Ki-67 genes or only detecting MCM2 and TOP2A genes by a single immunocytochemistry method wastes time and labor, two samples need to be processed simultaneously, then detection is carried out to obtain a result, the method is complex, a dyeing solution is easy to become old, the dyeing effect is unstable, the accuracy of pathological interpretation of experimenters is influenced, observation is inconvenient, the detection accuracy is low, the existing four-dyeing kit buffer solution needs to be divided into two test tubes for split charging when the loading amount is large, the pollution of the buffer solution is easy to cause, and the size of the kit can be increased if the loading amount of a single test tube is large.

SUMMERY OF THE UTILITY MODEL

In order to solve the problems in the background art, the present invention provides a detection kit for immunocytochemistry tetrastich, which has the advantages of improving the accuracy of pathological interpretation of experimenters, facilitating observation, improving the accuracy of detection, reducing the possibility of false positive and false negative, and reducing the possibility of erroneous judgment, and solves the problems of detection of only P16 and Ki-67 genes or only MCM2 and TOP2A genes by a single immunocytochemistry method, such as time and labor consumption, simultaneous processing of two samples, and detection to obtain results, which is complicated, easy obsolescence of staining solution, unstable staining effect, affecting the accuracy of pathological interpretation of experimenters, inconvenient observation, low accuracy of detection, and the existing buffer solution of tetrastich kit needs to be divided into two test tubes for subpackaging due to large loading amount, the buffer solution is easily polluted, and if the single test tube is filled in a large quantity, the size of the kit can be increased.

In order to achieve the above object, the utility model provides a following technical scheme: immunocytochemistry four-stain method detect reagent box, comprises a box bod, the inside of box is provided with the liner, the container hole has been seted up at the TOP of liner, the quantity of container hole is seven, the aseptic device hole that is located the container hole right side has been seted up at the TOP of liner, the quantity of aseptic device hole is four, the inside in container hole is provided with p16 one respectively and resists concentrate reagent bottle, Ki-67 one and resists concentrate reagent bottle, MCM2 one and resists concentrate reagent bottle, TOP2A one and resists concentrate reagent bottle, goat anti mouse two antibody working solution reagent bottle, goat anti rabbit two antibody working solution reagent bottle and DAB dyeing substrate reagent bottle, the inside in aseptic device hole is provided with antibody diluent and DAB dyeing buffer solution reagent bottle respectively, the quantity of DAB dyeing buffer solution reagent bottle is three and evenly arranged inside the aseptic device hole on antibody diluent right side, the utility model discloses a kit, including pad, box body, inner chamber, push-press structure, antibody diluent and DAB dyeing buffer solution reagent bottle, the inner chamber that communicates with aseptic device hole is seted up to the inside of pad, the front side of inner chamber and the front of box body communicate, the inside sliding connection of inner chamber has the push-press structure, the back that pushes-press the structure sets up to the arc, the surface of antibody diluent and DAB dyeing buffer solution reagent bottle all with push-press the back contact of structure, the rear side swing joint at box body top has the apron, apron and box body make.

As the utility model discloses it is preferred, push away and to have seted up the bearing groove according to the surface of structure, push away and to set up the spacing groove that is located the bearing groove bottom according to the surface of structure.

As the utility model discloses it is preferred, the right side of box body is provided with the locating lever, the left end of locating lever run through box body, liner and spacing groove in proper order and with liner fixed connection, spacing groove and locating lever sliding connection.

As the utility model discloses it is preferred, the rear side fixedly connected with pressure spring of inner chamber, one side that the inner chamber was kept away from to the pressure spring with push away the back fixed connection according to the structure.

As the utility model discloses preferred, the bottom of apron and the equal fixedly connected with magnet in front of box body, magnet inter attraction.

As the utility model discloses preferred, p16 anti concentrate reagent bottle, Ki-67 anti concentrate reagent bottle, MCM2 anti concentrate reagent bottle, TOP2A anti concentrate reagent bottle, sheep are anti two work solution reagent bottles in mouse, sheep are anti two work solution reagent bottles in rabbit and DAB dyeing substrate reagent bottle can place at will in the inside in container hole and distinguish through the colour.

Compared with the prior art, the beneficial effects of the utility model are as follows:

1. the utility model has compact structure, convenient carrying, good repeatability, time and labor saving, high efficiency and stability of immunocytochemistry four-staining detection of P16/Ki-67 and MCM2/TOP2A, can well meet the needs of science and work, solves the problems that the method of immunocytochemistry is used for single time to detect only P16 and Ki-67 genes or only MCM2 and TOP2A genes, wastes time and labor in detection, needs to process two samples simultaneously, then the detection is carried out to obtain a result, which is more complex, the staining solution is easy to be old, the staining effect is unstable, the accuracy of pathological interpretation of experimenters is influenced, the observation is inconvenient, the detection accuracy is low, and current four dye kit buffer solution need be divided into two test tubes and carry out the partial shipment because the dress volume is great, causes buffer solution's pollution easily, if the big problem that can increase the kit volume again of single test tube dress volume.

2. The utility model discloses a set up bearing groove and spacing groove, can reduce and push away the cost according to the structure, can improve simultaneously and push away the intensity according to the structure, avoid pushing away according to the structure and pressing the in-process deformation that appears.

3. The utility model discloses a set up the locating lever, can play spacing effect to pushing away according to the structure, avoid pushing away according to the structure and moving the in-process slope.

4. The utility model discloses a set up the pressure spring, can play the effect of support to pushing away according to the structure, push away and press the structure and can directly reset when breaking away from the extrusion, be convenient for push away the repetitious usage according to the structure.

5. The utility model discloses a set up magnet, can improve the connection effect of box body and apron, can fix the apron.

6. The utility model discloses a position and colour to the downthehole reagent bottle of container are injectd, can the person of facilitating the use distinguish being equipped with different types of reagent bottle, improve the installation effectiveness of reagent bottle.

Drawings

FIG. 1 is a schematic structural view of the present invention;

FIG. 2 is a schematic left-side sectional view of the local structure of the present invention;

fig. 3 is an enlarged schematic view of a portion a in fig. 1 according to the present invention.

In the figure: 1. a box body; 2. a liner; 3. a container aperture; 4. a sterile device aperture; 5. p16 reagent bottle for primary concentrated solution; 6. ki-67 primary antibody concentrated solution reagent bottle; 7. MCM2 primary concentrated solution reagent bottle; 8. TOP2A first-antibody concentrate reagent bottle; 9. a goat anti-mouse secondary antibody working solution reagent bottle; 10. a goat anti-rabbit secondary antibody working solution reagent bottle; 11. DAB staining substrate reagent bottle; 12. antibody diluent; 13. DAB staining buffer solution reagent bottles; 14. an inner cavity; 15. a push structure; 16. A cover plate; 17. a load bearing groove; 18. a limiting groove; 19. positioning a rod; 20. a pressure spring; 21. and a magnet.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.

As shown in fig. 1 to 3, the immunocytochemistry four-staining detection kit provided by the present invention comprises a kit body 1, a gasket 2 is disposed in the kit body 1, container holes 3 are disposed at the TOP of the gasket 2, the number of the container holes 3 is seven, aseptic device holes 4 located at the right side of the container holes 3 are disposed at the TOP of the gasket 2, the number of the aseptic device holes 4 is four, p 16-anti-concentrate reagent bottles 5, Ki-67-anti-concentrate reagent bottles 6, MCM 2-anti-concentrate reagent bottles 7, TOP 2A-anti-concentrate reagent bottles 8, goat anti-mouse second antibody working solution reagent bottles 9, goat anti-rabbit second antibody working solution reagent bottles 10 and DAB staining substrate reagent bottles 11 are disposed in the container holes 4, DAB antibody diluent 12 and staining buffer reagent bottles 13 are disposed in the aseptic device holes 4, DAB reagent bottles 13 are three and uniformly arranged in the aseptic device holes 4 at the right side of the DAB diluent 12, the inner chamber 14 that communicates with aseptic device hole 4 is seted up to the inside of liner 2, the front side of inner chamber 14 and the front intercommunication of box body 1, the inside sliding connection of inner chamber 14 has and pushes away according to structure 15, the back that pushes away according to structure 15 sets up to the arc, the surface of antibody diluent 12 and DAB staining buffer solution reagent bottle 13 all with push away according to the back contact of structure 15, the rear side swing joint at box body 1 top has apron 16, apron 16 makes for integrated into one piece with box body 1.

Referring to fig. 2, the surface of the pushing structure 15 is provided with a bearing groove 17, and the surface of the pushing structure 15 is provided with a limiting groove 18 located at the bottom of the bearing groove 17.

As a technical optimization scheme, through setting up bearing groove 17 and spacing groove 18, can reduce and push away the cost according to structure 15, can improve simultaneously and push away the intensity according to structure 15, avoid pushing away according to structure 15 and appearing warping pressing the in-process.

Referring to fig. 2, a positioning rod 19 is disposed on the right side of the box body 1, the left end of the positioning rod 19 penetrates through the box body 1, the gasket 2 and the limiting groove 18 in sequence and is fixedly connected with the gasket 2, and the limiting groove 18 is connected with the positioning rod 19 in a sliding manner.

As a technical optimization scheme of the utility model, through setting up locating lever 19, can play spacing effect to pushing structure 15, avoid pushing structure 15 and removing the in-process slope.

Referring to fig. 2, a compression spring 20 is fixedly connected to the rear side of the inner cavity 14, and a side of the compression spring 20 away from the inner cavity 14 is fixedly connected to the back of the pushing structure 15.

As a technical optimization scheme of the utility model, through setting up pressure spring 20, can push away the effect that presses structure 15 to play the support, push away and press structure 15 and can directly reset when breaking away from the extrusion, be convenient for push away the repetitious usage who presses structure 15.

Referring to fig. 1, a magnet 21 is fixedly connected to the bottom of the cover 16 and the front surface of the case 1, and the magnets 21 attract each other.

As the utility model discloses a technical optimization scheme through setting up magnet 21, can improve the effect of being connected of box body 1 and apron 16, can fix apron 16.

Referring to fig. 3, a p16 primary anti-concentrate reagent bottle 5, a Ki-67 primary anti-concentrate reagent bottle 6, an MCM2 primary anti-concentrate reagent bottle 7, a TOP2A primary anti-concentrate reagent bottle 8, a goat anti-mouse secondary antibody working solution reagent bottle 9, a goat anti-rabbit secondary antibody working solution reagent bottle 10, and a DAB staining substrate reagent bottle 11 can be freely placed inside the container well 3 and distinguished by color.

As the utility model discloses a technical optimization scheme is injectd through the position and the colour to 3 inside reagent bottles in container hole, can convenient to use person distinguish being equipped with different types of reagent bottle, improves the installation effectiveness of reagent bottle.

The utility model discloses a theory of operation and use flow:

when the device is used, a user pushes the push structure 15 backwards, the push structure 15 extrudes the antibody diluent 12 and the DAB staining buffer reagent bottle 13 through the inclined plane, and the front ends of the antibody diluent 12 and the DAB staining buffer reagent bottle 13 move to the outside of the sterile device hole 4.

1. Primary antibody incubation:

1.1 after the kit is opened, 1.85ml of antibody diluent, 100 mu l of p16 and Ki-67 primary antibody concentrated solution are mixed and added into 100 mu l of MCM2 and TOP2A primary antibody concentrated solution to prepare p16/Ki-67 and MCM2/TOP2A primary antibody working mixed solution, a new label is pasted, and the mixed solution is stored at the temperature of 2-8 ℃ in a dark place for later use.

1.2 dropping 50 μ l/piece of primary antibody working mixed solution, incubating in a wet box at 37 ℃ for 30min, rinsing with PBST for 3min × 3 times, and spin-drying moderately.

2. And (3) secondary antibody incubation:

2.1 after the kit is opened, adding 1ml of goat anti-mouse secondary antibody working solution into 1ml of goat anti-rabbit secondary antibody working solution to prepare goat anti-mouse/rabbit secondary antibody working mixed solution, pasting a new label, and storing in a dark place at 2-8 ℃ for later use.

2.2 dropwise adding 50 μ l/piece of secondary antibody working mixed solution, incubating at 37 deg.C for 15min, rinsing with PBST for 3min × 3 times, and spin-drying moderately.

3. DAB dyeing:

3.1 buffer removal:

3.2 preparing DAB working mixed liquid (according to the sample amount, taking a proper amount of DAB dyeing substrate and DAB dyeing agent buffer solution to mix uniformly), using the mixture immediately after preparation, using the mixture within 4h, and storing the mixture at room temperature (20-25 ℃) in a dark place.

3.2 dropwise adding 50 mu l/piece of DAB working mixed solution, incubating for 5-10 min in a wet box at room temperature (20-25 ℃), washing for 3min by running water, and spin-drying moderately (the piece cannot be dried).

4. red staining:

4.1 preparing red working mixed solution, namely preparing the red working mixed solution immediately, using the red working mixed solution within 30min, and storing the red working mixed solution at room temperature (20-25 ℃) in a dark place.

4.2 dripping 50 mu l/piece of red working mixed solution, incubating for 10-30 min in a wet box at room temperature (20-25 ℃), washing for 3min with running water, and properly drying.

5. Counterdyeing: and (3) performing counterstaining on the cell slices in hematoxylin dye at room temperature (20-25 ℃) for 1s, washing for 3min with running water, and drying.

6. Sealing: 1 drop of neutral quick-sealing tablet is added dropwise, a cover slip is added, and the result is evaluated under an optical microscope.

7. And (4) analyzing results:

the result analysis is carried out according to the staining condition of the cell nucleus and the cell pulp of the same monolayer of cells or the same cell cluster.

After P16/Ki-67 and MCM2/TOP2A antibodies are incubated, DAB and red dyes are quadruplicate-stained, the cell nucleus of the same monolayer cell or cell cluster is blue or red, and the cytoplasm is not stained; or the cell nucleus is brown and the cell cytoplasm is brown; or the cell nucleus shows red, and the cytoplasm is not stained, namely the result is judged to be negative.

After P16/Ki-67 and MCM2/TOP2A antibodies are incubated, DAB dye is singly dyed, the cell nucleus of a single layer cell or a cell cluster is brown, and cytoplasm is brown; and (3) singly dyeing the red dye, wherein the cell nucleus of a single-layer cell or a cell cluster is red, and the cytoplasm is not dyed, so that the positive result is judged.

In summary, the following steps: the immunocytochemistry four-staining method detection kit has the advantages of compact structure, convenient carrying, good repeatability, time and labor saving, the immunocytochemistry four-staining detection of P16/Ki-67 and MCM2/TOP2A is efficient and stable, the requirements of science and work can be well met, the problems that the method of single-pass immunocytochemistry only detects P16 and Ki-67 genes or only detects MCM2 and TOP2A genes is solved, the detection is time-consuming and labor-consuming, two samples need to be processed simultaneously, then the detection is carried out to obtain a result, which is more complex, the staining solution is easy to be old, the staining effect is unstable, the accuracy of pathological interpretation of experimenters is influenced, the observation is inconvenient, the detection accuracy is low, and current four dye kit buffer solution need be divided into two test tubes and carry out the partial shipment because the dress volume is great, causes buffer solution's pollution easily, if the big problem that can increase the kit volume again of single test tube dress volume.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (6)

1. Immunocytochemistry four-stain method detect reagent box, including box body (1), its characterized in that: the utility model discloses a kit, including box body (1), container hole (3) have been seted up at the inside of box body (1), container hole (3) have been seted up at the TOP of liner (2), the quantity of container hole (3) is seven, aseptic device hole (4) that are located container hole (3) right side have been seted up at the TOP of liner (2), aseptic device hole (4)'s quantity is four, the inside of container hole (3) is provided with one respectively p16 anti-concentrate reagent bottle (5), one Ki-67 anti-concentrate reagent bottle (6), MCM2 one anti-concentrate reagent bottle (7), TOP2A one anti-concentrate reagent bottle (8), goat anti-mouse two antibody working solution reagent bottle (9), goat anti-rabbit two antibody working solution reagent bottle (10) and DAB staining substrate reagent bottle (11), the inside of aseptic device hole (4) is provided with antibody diluent (12) and DAB staining buffer solution reagent bottle (13) respectively, the quantity of DAB dyeing buffer solution reagent bottle (13) is three and inside aseptic device hole (4) on antibody diluent (12) right side of align to grid, inner chamber (14) with aseptic device hole (4) intercommunication is seted up to the inside of liner (2), the front side of inner chamber (14) and the front intercommunication of box body (1), the inside sliding connection of inner chamber (14) has and pushes away according to structure (15), it sets up to the arc to push away according to the back of structure (15), the surface of antibody diluent (12) and DAB dyeing buffer solution reagent bottle (13) all with push away according to the back contact of structure (15), the rear side swing joint at box body (1) top has apron (16), apron (16) and box body (1) make for integrated into one piece.

2. The immunocytochemistry four-stain detection kit according to claim 1, characterized in that: the surface of the push structure (15) is provided with a bearing groove (17), and the surface of the push structure (15) is provided with a limiting groove (18) positioned at the bottom of the bearing groove (17).

3. The immunocytochemistry tetrastich detection kit according to claim 2, characterized in that: the right side of box body (1) is provided with locating lever (19), the left end of locating lever (19) run through box body (1), liner (2) and spacing groove (18) in proper order and with liner (2) fixed connection, spacing groove (18) and locating lever (19) sliding connection.

4. The immunocytochemistry four-stain detection kit according to claim 1, characterized in that: the rear side of the inner cavity (14) is fixedly connected with a pressure spring (20), and one side, far away from the inner cavity (14), of the pressure spring (20) is fixedly connected with the back of the pushing structure (15).

5. The immunocytochemistry four-stain detection kit according to claim 1, characterized in that: the bottom of apron (16) and the equal fixedly connected with magnet (21) in front of box body (1), magnet (21) inter attraction.

6. The immunocytochemistry four-stain detection kit according to claim 1, characterized in that: the p16 primary anti-concentrated solution reagent bottle (5), the Ki-67 primary anti-concentrated solution reagent bottle (6), the MCM2 primary anti-concentrated solution reagent bottle (7), the TOP2A primary anti-concentrated solution reagent bottle (8), the goat anti-mouse secondary antibody working solution reagent bottle (9), the goat anti-rabbit secondary antibody working solution reagent bottle (10) and the DAB staining substrate reagent bottle (11) can be randomly placed in the container hole (3) and distinguished through colors.

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