CN212514619U - P16MCM2 immunocytochemistry double-staining detection kit - Google Patents

Abstract

The utility model discloses a P16MCM2 immunocytochemistry double-dyeing method detect reagent box, including box body and support high platform, the laminating of box body inner wall has the liner, and the liner upper surface is provided with aseptic device hole, the liner left side distributes has first recess, and first recess one side distributes and have the second recess, second recess one side distributes and has the third recess, first recess right side distributes and has the fourth recess, and fourth recess one side distributes and has the fifth recess, fifth recess one side distributes and has the sixth recess, support high platform and set up in first recess upper end. The P16MCM2 immunocytochemistry double-staining method detection kit is provided with a first groove, and the result of the immunocytochemistry double-staining method of P16/MCM2 is displayed on the same sample slide, namely the result is displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is facilitated, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced.

Description

P16MCM2 immunocytochemistry double-staining detection kit

Technical Field

The utility model relates to an immune cell detection kit technical field specifically is P16MCM2 immune cell chemistry double staining method detect reagent box.

Background

The p16 gene is also called a multi-tumor cancer suppressor gene, and the expressed cancer suppressor protein p16 is a cell cycle progression negative regulatory protein. When the human papilloma virus is integrated and infects cervical epithelial cells, an oncogene E7 is introduced and activated, so that the expression of an oncogenic protein E7 is promoted, pRB is inhibited from being combined with E2F protein, and the proliferation of the cervical epithelial cells is promoted; when the cell is over-proliferated, a negative feedback regulation mechanism of the cell proliferation is activated, namely, the cancer suppressor gene p16 is activated to promote the expression of the cancer suppressor protein p16 and promote the combination of pRB and E2F protein, so that the over-proliferation of the cervical epithelial cells is inhibited.

The MCM2 gene is related to cell proliferation, its expression varies with different cell cycle phases, starts to express in G1 phase of cell cycle, increases in S phase and G2 phase, reaches peak in M phase, degrades or loses antigenic determinant rapidly in the later stage of cell mitosis, and does not express in G0 phase. The half-life period is short, so that the cell growth fraction is not easy to induce by growth factors, and the cell growth fraction can be used as an index for evaluating the cell growth fraction.

The existing detection kit detects only P16 gene or only MCM2 gene by immunocytochemistry single-staining detection, namely a single-pass immunocytochemistry method.

In the application of the P16MCM2 immune cell detection kit in the market, in cervical cancer screening, only the P16 gene or only the MCM2 gene is detected by a single-pass immune cytochemistry method, the detection is time-consuming and labor-consuming, two samples need to be processed simultaneously, and then the detection is carried out to obtain a result. The method is complex, the staining solution is easy to become old, the staining effect is unstable, the accuracy of pathological interpretation of experimenters is affected, observation is inconvenient, and the detection accuracy is low. Two single stains are laborious and laborious, but if two stains are combined, a large number of reagents are required to be assembled in the same kit. The kit structure needs to be perfect. In the same double-staining kit, the antibody concentrated solution is divided into smaller tubes due to the small loading amount, and when the antibody concentrated solution is loaded into a large tube, the reagent is lost due to overturning and reversing in the transportation process, and therefore, the P16MCM2 immunocytochemistry double-staining detection kit is proposed.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide P16MCM2 immunocytochemistry double staining method detect reagent box to in solving and proposing in the above-mentioned background art in the cervical carcinoma screening, the method of single through immunocytochemistry only detects P16 gene or only detects the method of MCM2 gene, detects and wastes time and energy, needs two samples of simultaneous processing, then detects and obtains the result. The method is complex, the staining solution is easy to become old, the staining effect is unstable, the accuracy of pathological interpretation of experimenters is affected, observation is inconvenient, and the detection accuracy is low. Two single stains are laborious and laborious, but if two stains are combined, a large number of reagents are required to be assembled in the same kit. The kit structure needs to be perfect. In the same double-staining kit, the antibody concentrate is packed in smaller tubes due to its small amount, and when it is packed in large tubes, it is turned over and upside down during transportation, which causes a problem of loss of the reagent.

In order to achieve the above object, the utility model provides a following technical scheme: p16MCM2 immunocytochemistry double-staining method detect reagent box, including box body and support high platform, the laminating of box body inner wall has the liner, and the liner upper surface is provided with aseptic device hole, the liner left side distributes and has first recess, and first recess one side distributes and has the second recess, second recess one side distributes and has the third recess, first recess right side distributes and has the fourth recess, and fourth recess one side distributes and has the fifth recess, fifth recess one side distributes and has the sixth recess, support high platform sets up in first recess upper end, the downthehole aseptic device body that distributes of aseptic device.

Preferably, the inner wall of the box body is attached to the outer wall of the liner, and the vertical central axis of the box body coincides with the vertical central axis of the liner.

Preferably, the sterile device holes are symmetrically distributed about a horizontal central axis of the pad, and the depth dimension of the sterile device holes is smaller than the thickness dimension of the pad.

Preferably, the first grooves and the gasket are distributed vertically, and the first grooves and the third grooves are distributed symmetrically about a horizontal central axis of the gasket.

Preferably, the elevating platform is symmetrically distributed about the vertical central axis of the first groove, and the elevating platform coincides with the vertical central axis of the first groove.

Preferably, the size of the inner opening of the sterile device hole is matched with the size of the outer opening of the sterile device body, and the depth size of the sterile device hole is larger than the width size of the sterile device body.

Compared with the prior art, the beneficial effects of the utility model are that: the P16MCM2 immunocytochemistry double-staining method detection kit is provided with a first groove, the first groove and a third groove are symmetrically distributed about a horizontal central axis of a gasket, DAB staining buffer solution reagent bottles are respectively placed in the first groove, the second groove and the third groove, P16 anti-concentrated solution, MCM2 anti-concentrated solution and antibody diluent are respectively contained in the DAB staining buffer solution reagent bottles, and the results of the P16/MCM2 immunocytochemistry double-staining method are displayed on the same sample slide, namely, the results are displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is facilitated, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced.

The depth dimension in aseptic device hole is less than the size of liner, and the setting of liner plays the guard action to various reagent bottles and aseptic device bodies of box body inside, alleviates the impact that the box body rocked time and to reagent bottle and aseptic device body production, protects the safety of reagent bottle and aseptic device body, satisfies user's demand.

The depth dimension in aseptic device hole is greater than the width dimension of aseptic device body, lays the great buffer solution of volume in the aseptic device hole, so the aseptic device body adopts transversely to lay for the device atress is more reasonable, and the aseptic device body is more stable.

Drawings

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

FIG. 2 is a schematic view of the cross-sectional structure of the first groove of the present invention;

FIG. 3 is a schematic view of the cross-sectional structure of a fourth groove of the present invention;

fig. 4 is a schematic view of the pore structure of the aseptic device of the present invention.

In the figure: 1. a box body; 2. a liner; 3. a sterile device aperture; 4. a first groove; 5. a second groove; 6. a third groove; 7. a fourth groove; 8. a fifth groove; 9. a sixth groove; 10. a lifting platform; 11. a sterile device body.

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.

Referring to fig. 1-4, the present invention provides a technical solution: the P16MCM2 immunocytochemistry double-staining detection kit comprises a kit body 1, a gasket 2, a sterile device hole 3, a first groove 4, a second groove 5, a third groove 6, a fourth groove 7, a fifth groove 8, a sixth groove 9, a height support table 10 and a sterile device body 11, wherein the gasket 2 is attached to the inner wall of the kit body 1, the inner wall of the kit body 1 is attached to the outer wall of the gasket 2, the vertical central axis of the kit body 1 is overlapped with the vertical central axis of the gasket 2, the sterile device hole 3 is arranged on the upper surface of the gasket 2, the sterile device hole 3 is symmetrically distributed relative to the horizontal central axis of the gasket 2, the depth of the sterile device hole 3 is smaller than the thickness of the gasket 2, the gasket 2 is arranged to protect various reagent bottles and the sterile device body 11 in the kit body 1, impact on the reagent bottles and the sterile device body 11 when the kit body 1 shakes is reduced, and the safety of the reagent bottles and the sterile device body 11 is protected, the requirements of users are met;

a first groove 4 is distributed on the left side of the gasket 2, a second groove 5 is distributed on one side of the first groove 4, a third groove 6 is distributed on one side of the second groove 5, the first groove 4 and the gasket 2 are distributed in a vertical shape, the first groove 4 and the third groove 6 are symmetrically distributed about the horizontal central axis of the gasket 2, DAB staining buffer solution reagent bottles are respectively arranged in the first groove 4, the second groove 5 and the third groove 6, and the DAB staining buffer solution reagent bottle is respectively filled with P16 primary anti-concentrated solution, MCM2 primary anti-concentrated solution and antibody diluent, the result of the immunocytochemistry double staining method of P16/MCM2 is displayed on the same sample slide, the result is displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is facilitated, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced;

a fourth groove 7 is distributed on the right side of the first groove 4, a fifth groove 8 is distributed on one side of the fourth groove 7, a sixth groove 9 is distributed on one side of the fifth groove 8, a height supporting table 10 is arranged at the upper end of the first groove 4, the height supporting tables 10 are symmetrically distributed about the vertical central axis of the first groove 4, the height supporting tables 10 are superposed with the vertical central axis of the first groove 4, DAB staining buffer solution reagent bottles are respectively placed in the fourth groove 7, the fifth groove 8 and the sixth groove 9, and goat anti-mouse secondary antibody working solution, goat anti-rabbit secondary antibody working solution and DAB staining substrates are respectively contained in the DAB staining buffer solution bottles, and the reagent bottles can be prevented from overturning due to the fact that the buffer solutions placed in the first groove 4 and the second groove 5 are small in size and the height supporting tables 10 are used as a padding height design;

aseptic device body 11 has been distributed in aseptic device hole 3, and the internal orifice size in aseptic device hole 3 is identical with the external orifice size of aseptic device body 11, and the degree of depth size in aseptic device hole 3 is greater than aseptic device body 11's width size, lays the great buffer solution of volume in the aseptic device hole 3, so aseptic device body 11 adopts transversely to lay for the device atress is more reasonable, and aseptic device body 11 is more stable.

The working principle is as follows: for the detection kit of the P16MCM2 immunocytochemistry double-staining method, the arrangement of the liner 2 plays a role in protecting various reagent bottles and the sterile device body 11 in the box body 1, reduces the impact on the reagent bottles and the sterile device body 11 when the box body 1 shakes, protects the safety of the reagent bottles and the sterile device body 11, and meets the requirements of users, because the buffer solution placed in the first groove 4 and the second groove 5 has smaller volume, the elevating platform 10 is adopted as a cushion height design, the reagent bottles can be prevented from overturning, and the buffer solution with larger volume is placed in the sterile device hole 3, the sterile device body 11 is placed horizontally, so that the stress of the device is more reasonable, and the sterile device body 11 is more stable;

the immunocytochemistry detection method comprises seven steps, namely primary antibody incubation, secondary antibody incubation, DAB staining, red staining, counterstaining, mounting and result analysis;

firstly, primary anti-incubation is carried out, after a box body 1 is opened, reagent bottles are taken out from a first groove 4, a second groove 5 and a third groove 6, 1.85ml of antibody diluent and 100 mu l p16 primary anti-concentrated solution are mixed and added into 100 mu l MCM2 primary anti-concentrated solution to prepare p16/l MCM2 primary anti-working mixed solution, a new label is pasted, the mixture is stored in a dark place at the temperature of 2-8 ℃ for later use, the reagent bottles are taken out from a fourth groove 7, a fifth groove 8 and a sixth groove 9, 50 mu l/piece of the primary anti-working mixed solution is dripped, wet box incubation is carried out for 30min at the temperature of 37 ℃, PBST is rinsed for 3min multiplied by 3 times, and the reagent bottles are properly dried;

then, performing secondary antibody incubation, after the box body 1 is opened, taking out a reagent bottle from the fourth groove 7, the fifth groove 8 and the sixth groove 9, adding 1ml of goat-anti-mouse secondary antibody working solution into 1ml of goat-anti-rabbit secondary antibody working solution to prepare a goat-anti-mouse/rabbit secondary antibody working mixed solution, pasting a new label, storing in a dark place at 2-8 ℃ for later use, dropwise adding 50 mu l/piece of the secondary antibody working mixed solution, incubating in a wet box at 37 ℃ for 15min, rinsing PBST for 3min × 3 times, and spin-drying appropriately;

performing DAB staining, taking out a buffer solution, preparing DAB working mixed solution (according to the sample amount, taking a proper amount of DAB staining substrate and DAB staining agent buffer solution and mixing uniformly), using the DAB working mixed solution immediately after preparation, using the DAB working mixed solution within 4h, storing the DAB working mixed solution at room temperature (20-25 ℃) in a dark place, dropwise adding 50 mu l/piece of DAB working mixed solution, incubating the DAB working mixed solution in a wet box at room temperature (20-25 ℃) for 5-10 min, washing the DAB working mixed solution for 3min with;

performing red dyeing, preparing red working mixed liquid, namely preparing the red working mixed liquid, namely using the red working mixed liquid, using the red working mixed liquid within 30min, storing the red working mixed liquid at room temperature (20-25 ℃) in a dark place, dripping 50 mu l/piece of the red working mixed liquid, incubating the red working mixed liquid in a wet box at room temperature (20-25 ℃) for 10-30 min, flushing the red working mixed liquid for 3min with running water, properly spin-drying the red working mixed liquid, and finishing red dyeing;

counterstaining is carried out, cell slices are placed into hematoxylin dye for dip staining, counterstaining is carried out for 1s at room temperature (20-25 ℃), washing is carried out for 3min, drying is carried out, then, blocking is carried out, 1 drop of neutral rapid blocking tablet is dripped, a cover glass is covered, and the result is evaluated under an optical microscope;

finally, performing result analysis, wherein the result analysis is performed according to the dyeing condition of the cell nucleus and the cell pulp of the same single-layer cell or the same cell cluster, after the P16/MCM2 antibody is incubated, DAB and red dyes are doubly dyed, the cell nucleus of the same single-layer cell or the cell cluster is blue or red, and the cell pulp is not dyed; or the cell nucleus is brown and the cell cytoplasm is brown; or the cell nucleus is red, the cytoplasm is not stained, namely a negative result is judged, after the incubation of the P16/MCM2 antibody, the DAB dye is singly stained, the cell nucleus of a single-layer cell or a cell cluster is brown, and the 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.

The immunocytochemistry double-staining of P16/MCM2, in the staining process, the primary antibodies of two proteins are mixed and then incubated to obtain a sample, and meanwhile, the expression of the two proteins is detected on a sample slide, so that the method is cheap and fast and can almost increase the detection speed by one time on the premise of accurately screening the early cervical cancer. The kit is superior to similar immunocytochemistry single-stain kits for detecting P16 gene or only MCM2 gene. The result of the immunocytochemistry double-staining method of P16/MCM2 is displayed on the same sample slide, namely the result is displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is convenient, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced.

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. The P16MCM2 immunocytochemistry double-staining detection kit comprises a kit body (1) and a height supporting platform (10), and is characterized in that: the utility model discloses a box body, including box body (1), box body, pad (2), and pad (2) upper surface are provided with aseptic device hole (3), pad (2) the left side distributes and has first recess (4), and first recess (4) one side distributes and have second recess (5), second recess (5) one side distributes and has third recess (6), first recess (4) right side distributes and has fourth recess (7), and fourth recess (7) one side distributes and has fifth recess (8), fifth recess (8) one side distributes and has sixth recess (9), ask high platform (10) to set up in first recess (4) upper end, it has aseptic device body (11) to distribute in aseptic device hole (3).
2. 2. The P16MCM2 immunocytochemistry double stain detection kit of claim 1, wherein: the inner wall of the box body (1) is attached to the outer wall of the gasket (2), and the vertical central axis of the box body (1) coincides with the vertical central axis of the gasket (2).
3. 3. The P16MCM2 immunocytochemistry double stain detection kit of claim 1, wherein: the sterile device holes (3) are symmetrically distributed about the horizontal central axis of the pad (2), and the depth dimension of the sterile device holes (3) is smaller than the thickness dimension of the pad (2).
4. 4. The P16MCM2 immunocytochemistry double stain detection kit of claim 1, wherein: the first grooves (4) and the gasket (2) are distributed vertically, and the first grooves (4) and the third grooves (6) are symmetrically distributed about a horizontal central axis of the gasket (2).
5. 5. The P16MCM2 immunocytochemistry double stain detection kit of claim 1, wherein: the elevating platform (10) is symmetrically distributed about the vertical central axis of the first groove (4), and the elevating platform (10) is superposed with the vertical central axis of the first groove (4).
6. 6. The P16MCM2 immunocytochemistry double stain detection kit of claim 1, wherein: the size of the inner opening of the sterile device hole (3) is matched with the size of the outer opening of the sterile device body (11), and the depth size of the sterile device hole (3) is larger than the width size of the sterile device body (11).

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