A rapid iterative tsa multiplex immunofluorescence staining kit

Abstract

The application discloses a rapid iteration type TSA multiple immunofluorescence staining kit, relates to the technical field of multiple immunofluorescence staining, and comprises an operation table, an elution tank is installed on the outer wall of the operation table, a shaking-off clamp is installed on the inner wall left side of the elution tank, the shaking-off clamp is connected with the elution tank through a rotating shaft one, a dropwise adding device is installed on the outer wall rear side of the elution tank, the dropwise adding device is connected with the operation table through a moving assembly, a liquid storage tank one is installed on the inner wall rear side of the operation table, a pressure pump one is installed on the outer wall upper side of the liquid storage tank one, and the dropwise adding device is connected with the pressure pump one through a connecting pipe. Through the installation of the shaking-off clamp, the dropwise adding device, the moving assembly and the rotating shaft two, the function of precise dropwise adding of eluent is realized, the problems of fixed eluent dropwise adding parameters, slice tissue damage, uneven eluent coverage and cross contamination are solved, fragile samples can be protected, and the consistency of elution effect is improved.

Description

Technical Field

[0001] This invention relates to the field of multiplex immunofluorescence staining technology, specifically to a rapid iterative TSA multiplex immunofluorescence staining kit. Background Technology

[0002] Traditional immunohistochemistry relies on chromogenic substrates and can only detect 1-2 biomarkers per test. It also suffers from signal superposition interference and low-abundance target misses, which cannot meet the complex analytical needs of multi-cell interaction studies such as the tumor immune microenvironment. TSA (tyramine signal amplification) technology uses the tyramine signal amplification mechanism and HRP (horseradish peroxidase) to catalyze the covalent deposition of fluorescently labeled tyramine at the antigen site, thereby achieving geometric signal amplification and improving detection sensitivity and multi-target compatibility. During the iteration process, it is necessary to select the correct elution buffer and ensure the elution effect. This is to avoid incompletely eluted high-affinity antibodies binding to secondary antibodies in subsequent rounds, generating fluorescence signals in non-target areas, leading to false positive results. Elution residues cause fluorescein to deposit at non-target antigen sites, destroying the true spatial distribution information of target molecules. Residual signals increase non-specific background, reduce the signal-to-noise ratio, and may mask the weak signals of low-abundance targets.

[0003] Patent CN118010456B discloses a multiplex immunofluorescence staining method and kit for detecting mismatch repair proteins. The above patent enables simpler, more efficient and accurate detection of MMR status, thereby more accurately screening the population that can benefit from immunotherapy.

[0004] The aforementioned patent describes a process involving the preparation of primary antibody and dye working solutions for mismatch repair proteins. The sample to be stained, the primary antibody working solution, the dye working solution, the secondary antibody reagent, the antigen retrieval solution, the peroxidase blocking agent, and the washing solution are placed in their respective positions on a staining machine. The staining program is then run, and the four mismatch repair proteins are stained sequentially in the order of MSH6, MLH1, PMS2, and MSH2 to obtain stained sample sections. These sections are then counterstained with a fluorescence-quenching mounting medium and mounted to obtain the test sample. After imaging and software analysis, analytical data for the four mismatch repair proteins can be obtained, thus enabling a simpler, more efficient, and accurate detection of MMR status. There is room for optimization in terms of section elution uniformity.

[0005] Therefore, this application proposes a rapid iterative TSA multiplex immunofluorescence staining kit for precise addition of elution buffer. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid iterative TSA multiplex immunofluorescence staining kit to solve the technical problems mentioned in the background art, such as slide damage and distorted detection results caused by improper slide elution.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a rapid iterative TSA multiplex immunofluorescence staining kit, comprising an operating table, an elution tank mounted on the upper side of the outer wall of the operating table, a sling clip mounted on the left side of the inner wall of the elution tank, the sling clip being connected to the elution tank via a rotating shaft, a dropper mounted on the rear side of the outer wall of the elution tank, the dropper being connected to the operating table via a moving component, a storage tank mounted on the rear side of the inner wall of the operating table, a pressure pump mounted on the upper side of the outer wall of the storage tank, the dropper being connected to the pressure pump via a connecting pipe, an elution motor mounted on the left side of the outer wall of the pressure pump, the sling clip, the rotating shaft, the moving component, and the pressure pump being respectively connected to a switch at the output end of the elution motor via connecting shafts, and the elution motor being connected to a controller mounted on the front side of the outer wall of the operating table via a signal line.

[0008] Preferably, the moving component consists of a telescopic shaft and a rotating shaft II. The telescopic shaft is installed on the rear side of the outer wall of the elution tank, and the rotating shaft II is installed on the front side of the outer wall of the telescopic shaft. The telescopic shaft and the rotating shaft II are respectively connected to the elution motor via connecting shafts. A scanner is installed on the front side of the outer wall of the moving component. The scanner is connected to the controller via a signal line. Heating wires are installed on both the front and rear sides of the inner wall of the first storage tank. A temperature sensor I is installed on the rear side of the inner wall of the first storage tank. The heating wires are connected to the temperature sensor I via a signal line.

[0009] Preferably, a second liquid storage tank and a third liquid storage tank are installed on the left and right sides of the inner wall of the operating table, respectively. A second pressure pump is installed on the outer wall of the second liquid storage tank near the elution tank. The second pressure pump is connected to the inlet valve installed on the left side of the inner wall of the elution tank through a connecting pipe. The second liquid storage tank is connected to the drain valve installed on the lower side of the inner wall of the elution tank through a connecting pipe. A liquid level sensor is embedded on the upper side of the outer wall of the elution clamp close to the inner wall of the elution tank. A replacement motor is installed on the lower side of the outer wall of the second pressure pump. The second pressure pump is connected to the switch at the output end of the replacement motor through a connecting shaft. The inlet valve, the drain valve, the liquid level sensor, and the replacement motor are all connected to the controller through signal lines.

[0010] Preferably, a telescopic plate one is provided on the lower side of the outer wall of the elution clamp, and a telescopic plate two is provided on the upper side of the outer wall of the liquid level sensor. Both telescopic plates one and two are fixedly connected to the rear side of the inner wall of the elution tank. Both telescopic plates one and two are provided with sealing rings on their outer walls. A closed motor is installed on the lower side of the outer wall of the elution tank. Both telescopic plates one and two are connected to the switch at the output end of the closed motor through connecting shafts. Both telescopic plates one and two are provided with leakage valves through their surfaces. Both the closed motor and the leakage valves are connected to the controller through signal lines.

[0011] Preferably, a reagent box is installed on the upper side of the outer wall of the second liquid storage tank, and a slice box is installed on the upper side of the outer wall of the third liquid storage tank. An opening and closing plate is installed at the connection between the reagent box and the slice box and the operating table. A partition is provided in the middle of the inner wall of the reagent box. A sliding groove is provided on the lower side of the inner wall of the reagent box. A slider is provided in the middle of the inner wall of the sliding groove. A push plate is installed on the upper side of the outer wall of the slider. The opening and closing plate and the slider are respectively connected to the switch at the output end of the closed motor through a connecting shaft.

[0012] Preferably, the dripper consists of a drip inlet, a regulating valve, and a liquid storage chamber. The liquid storage chamber is installed on the lower side of the outer wall of the rotating shaft, and the drip inlet is installed on the lower side of the outer wall of the liquid storage chamber. A regulating valve is installed at the connection between the drip inlet and the liquid storage chamber. The liquid storage chamber is connected to a pressure pump through a connecting pipe, and the regulating valve is connected to a controller through a signal line.

[0013] Preferably, a partition plate 2 is provided in the middle of the inner wall of the liquid storage tank 2, and a refrigeration unit is embedded in both the inner wall of the liquid storage tank 2 and the inner wall of the partition plate 2. A temperature sensor 2 is installed on the lower side of the inner wall of the liquid storage tank 2, and a switching valve is installed at the connection between the liquid storage tank 2 and the pressure pump 2. The refrigeration unit, the temperature sensor 2, and the switching valve are all connected to the controller through signal lines.

[0014] Preferably, the reagent box and the slide box are equipped with an insulation layer inside, and a circulation pump is installed on the upper side of the outer wall of the pressure pump. The insulation layer is connected to the circulation pump through a connecting pipe, and the circulation pump is connected to the switch at the output end of the change motor through a connecting shaft.

[0015] Preferably, a weight sensor is installed at the connection between the detachment clamp and the rotating shaft, and the weight sensor is connected to the controller via a signal line.

[0016] Preferably, an infrared sensor is embedded on the left side of the inner wall of the elution tank at the same height as the elution clamp, and the infrared sensor is connected to the controller via a signal line.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention, by installing a elution clamp, a dropper, a moving component, and a rotating shaft, achieves the function of precise addition of elution solution, solves the problems of fixed elution solution addition parameters, tissue damage, uneven elution solution coverage, and cross-contamination, protects fragile samples, and improves the consistency of elution effect; 2. This invention, through the installation of a storage tank II, a storage tank III, and an elution pool, achieves the function of rapid cleaning of slides, solves the problems of residual elution solution, sample damage, and deviation in detection results, avoids fluorescence signal interference, improves detection accuracy, and reduces the risk of tissue detachment; 3. This invention, through the installation of a telescopic plate I, a telescopic plate II, and a liquid storage tank I, achieves the function of maintaining the slide incubation environment, solves the problem of slide damage caused by unstable incubation environment interfering with antibody binding and elution, avoids environmental changes interfering with slide incubation, improves detection accuracy, and reduces slide damage; 4. This invention, by incorporating a reagent box, a slide box, and a circulation pump, achieves precise reagent delivery, solves the problems of incomplete elution and uneven elution coverage caused by mixing primary and secondary antibodies, eliminates human error, and improves detection repeatability, accuracy, and efficiency. Attached Figure Description

[0018] Figure 1 This is a front view structural diagram of the present invention; Figure 2 This is a side view of the present invention; Figure 3 This is a schematic diagram of the connection structure between the liquid storage tank 2 and the liquid storage tank 3 and the elution tank of the present invention; Figure 4 This is a schematic diagram of the structure of telescopic plate one and telescopic plate two of the present invention; Figure 5 This is a schematic diagram of the reagent box structure of the present invention; Figure 6 This is a schematic diagram of the dropper structure of the present invention; Figure 7 This is a schematic diagram of the connection structure between the reagent box, the slide box, and the storage tank of the present invention; Figure 8 This is a schematic diagram of the structure of the detachment clamp and rotating shaft of the present invention.

[0019] In the diagram: 1. Operating table; 2. Washing tank; 3. Evaporation clamp; 4. Rotating shaft one; 5. Dropper; 6. Storage tank one; 7. Pressure pump one; 8. Washing motor; 9. Controller; 10. Telescopic shaft; 11. Rotating shaft two; 12. Scanner; 13. Heating wire; 14. Temperature sensor one; 15. Storage tank two; 16. Storage tank three; 17. Pressure pump two; 18. Inlet valve; 19. Drain valve; 20. Liquid level sensor; 21. Replacement motor; 22. Telescopic plate one; 23. Telescopic plate II; 24. Sealing ring; 25. Enclosed motor; 26. Leakage valve; 27. Reagent box; 28. Slice box; 29. ​​Opening and closing plate; 30. Partition I; 31. Slide groove; 32. Sliding block; 33. Push plate; 34. Dropping port; 35. Regulating valve; 36. Liquid storage chamber; 37. Partition II; 38. Refrigeration unit; 39. Temperature sensor II; 40. Switching valve; 41. Insulation jacket; 42. Circulation pump; 43. Weight sensor; 44. Infrared sensor. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0022] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0023] Example 1: Please refer to Figure 1 , Figure 2 , Figure 5 and Figure 8 A rapid iterative TSA multiplex immunofluorescence staining kit includes an operating table 1. An elution tank 2 is installed on the upper side of the outer wall of the operating table 1. A spitting clip 3 is installed on the left side of the inner wall of the elution tank 2. The spitting clip 3 is connected to the elution tank 2 via a rotating shaft 4. A dropper 5 is installed on the rear side of the outer wall of the elution tank 2. The dropper 5 is connected to the operating table 1 via a moving component. A storage tank 6 is installed on the rear side of the inner wall of the operating table 1. A pressure pump 7 is installed on the upper side of the outer wall of the storage tank 6. The dropper 5 is connected to the pressure pump 7 via a connecting pipe. An elution motor 8 is installed on the left side of the outer wall of the pressure pump 7. The spitting clip 3, rotating shaft 4, moving component, and pressure pump 7 are respectively connected to a switch at the output end of the elution motor 8 via connecting shafts. The elution motor 8 is connected to a controller 9 installed on the front side of the outer wall of the operating table 1 via a signal line. The moving assembly consists of a telescopic shaft 10 and a rotating shaft 11. The telescopic shaft 10 is installed on the rear side of the outer wall of the elution tank 2, and the rotating shaft 11 is installed on the front side of the outer wall of the telescopic shaft 10. The telescopic shaft 10 and the rotating shaft 11 are respectively connected to the elution motor 8 through connecting shafts. A scanner 12 is installed on the front side of the outer wall of the moving assembly. The scanner 12 is connected to the controller 9 through a signal line. Heating wires 13 are installed on both the front and rear sides of the inner wall of the storage tank 6. A temperature sensor 14 is installed on the rear side of the inner wall of the storage tank 6. The heating wires 13 are connected to the temperature sensor 14 through a signal line. The dropper 5 consists of a dropper port 34, a regulating valve 35, and a liquid storage chamber 36. The liquid storage chamber 36 is installed on the lower side of the outer wall of the rotating shaft 11. The dropper port 34 is installed on the lower side of the outer wall of the liquid storage chamber 36. The regulating valve 35 is installed at the connection between the dropper port 34 and the liquid storage chamber 36. The liquid storage chamber 36 is connected to the pressure pump 7 through a connecting pipe. The regulating valve 35 is connected to the controller 9 through a signal line. A weight sensor 43 is installed at the connection between the detachment clip 3 and the rotating shaft 4. The weight sensor 43 is connected to the controller 9 via a signal line. An infrared sensor 44 is embedded in the left side of the inner wall of the washing tank 2 at the same height as the detachment clamp 3. The infrared sensor 44 is connected to the controller 9 through a signal line. Further, the operator places the pre-processed slices in front of the elution clamp 3. The controller 9 controls the switch at the output of the elution motor 8 to connect to the elution clamp 3. Driven by the elution motor 8, the elution clamp 3 holds and fixes the slices. The controller 9 controls the heating wire 13 to connect to an external power source to heat the eluent stored in the storage tank 6. Heating stops when the temperature sensor 14 inside the storage tank 6 detects that the eluent temperature has risen to 37°C. The controller 9 then controls the switch at the output of the elution motor 8 to connect to the pressure pump 7. Driven by the elution motor 8, the 37°C eluent stored in the storage tank 6 is pressurized and delivered to the storage chamber 36 of the dropper 5 for temporary storage. Subsequently, the controller 9 controls the regulating valve 35 to adjust the flow rate at the dropper 3. The flow rate of the elution motor 8 is adjusted, and the elution motor 8 is controlled to synchronously deliver power to the moving component to adjust the position of the dropper 5. Different types of sections require different elution liquid drop heights. For example, frozen sections need to be completely covered with elution liquid, but if the elution liquid drop height is too high, the surface tension of the liquid can easily cause tissue detachment or accelerate tissue peeling. Paraffin sections are fixed in formaldehyde and embedded in paraffin, and can tolerate slightly higher drop heights, but overflow must be prevented to avoid cross-contamination. Therefore, the controller 9 determines the type of section based on the scanning results of the section held by the elution clamp 3 by the scanner 12. According to the type of section, the controller 9 controls the switch at the output end of the elution motor 8 to connect the telescopic shaft 10 and the rotating shaft 11 in sequence. Under the drive of the elution motor 8, the telescopic shaft 10... The height of the dropper 5 is adjusted to ensure the eluent falls from different heights, preventing damage to the tissue sample from excessive drop height or incomplete coverage of the section surface from insufficient drop height. The angle of the dropper 5 is adjusted via the rotating shaft 11 to ensure even distribution of the eluent on the section surface. After eluent addition, the scanner 12 scans the section to obtain the distribution of the eluent on the section surface. If the eluent does not completely cover the section surface due to tissue adsorption, the controller 9 controls the elution motor 8 to power the rotating shaft 11. Driven by the elution motor 8, the rotating shaft 11 adjusts the angle of the section using the elution clamp 3, ensuring the eluent is evenly distributed across the section surface. During the temperature adjustment process, the eluent can be evenly distributed on the slice surface, thereby ensuring the elution effect of the eluent on the slice. After the eluent is dropped onto the slice, the weight sensor 43 collects the weight of the slice held by the elution clamp 3 to ensure that the eluent is not over-added. That is, when the weight sensor 43 detects that the weight of the slice held by the elution clamp 3 has increased to the set value, the dripping of the dripper 5 is stopped evenly, even if the eluent coverage on the slice surface is uneven. At this time, the angle of the elution clamp 3 is adjusted by rotating shaft 4 to make the eluent evenly cover the slice. At the same time, the infrared sensor 44 collects information on the thickness of the eluent covering the slice surface to ensure that the eluent coverage thickness meets the requirements. The eluent thickness on the surface of frozen slices is 1-2 mm, and the eluent thickness on the surface of paraffin slices is 2-3 mm.

[0024] Example 2: Please refer to Figure 1 , Figure 3 and Figure 7 A rapid iterative TSA multiplex immunofluorescence staining kit includes an operating table 1. An elution tank 2 is installed on the upper side of the outer wall of the operating table 1. A spitting clip 3 is installed on the left side of the inner wall of the elution tank 2. The spitting clip 3 is connected to the elution tank 2 via a rotating shaft 4. A dropper 5 is installed on the rear side of the outer wall of the elution tank 2. The dropper 5 is connected to the operating table 1 via a moving component. A storage tank 6 is installed on the rear side of the inner wall of the operating table 1. A pressure pump 7 is installed on the upper side of the outer wall of the storage tank 6. The dropper 5 is connected to the pressure pump 7 via a connecting pipe. An elution motor 8 is installed on the left side of the outer wall of the pressure pump 7. The spitting clip 3, rotating shaft 4, moving component, and pressure pump 7 are respectively connected to a switch at the output end of the elution motor 8 via connecting shafts. The elution motor 8 is connected to a controller 9 installed on the front side of the outer wall of the operating table 1 via a signal line. The operating table 1 has a liquid storage tank 2 15 and a liquid storage tank 3 16 installed on the left and right sides of the inner wall, respectively. The outer wall of the liquid storage tank 2 15 is equipped with a pressure pump 2 17 near the washing tank 2. The pressure pump 2 17 is connected to the water inlet valve 18 installed on the left side of the inner wall of the washing tank 2 through a connecting pipe. The liquid storage tank 2 15 is connected to the drain valve 19 installed on the lower side of the inner wall of the washing tank 2 through a connecting pipe. The upper side of the outer wall of the detachment clamp 3 is embedded with a liquid level sensor 20 close to the inner wall of the washing tank 2. The lower side of the outer wall of the pressure pump 2 17 is equipped with a replacement motor 21. The pressure pump 2 17 is connected to the switch at the output end of the replacement motor 21 through a connecting shaft. The water inlet valve 18, the drain valve 19, the liquid level sensor 20 and the replacement motor 21 are all connected to the controller 9 through signal lines. A partition 37 is provided in the middle of the inner wall of the liquid storage tank 15. A refrigeration unit 38 is embedded in the inner walls of both the liquid storage tank 15 and the partition 37. A temperature sensor 39 is installed on the lower side of the inner wall of the liquid storage tank 15. A switching valve 40 is installed at the connection between the liquid storage tank 15 and the pressurizing pump 17. The refrigeration unit 38, the temperature sensor 39 and the switching valve 40 are all connected to the controller 9 through signal lines. Furthermore, the controller 9 records the time it takes for the elution solution to cover the slice surface. For example, the first elution solution coverage for frozen slices only requires 10 seconds, while the second elution solution coverage requires 5-20 minutes. Depending on the slice type, after the set time is reached, the controller 9 controls the elution motor 8 to drive the rotating shaft 4 to flip the elution clamp 3, causing the slice to rotate 180°. The elution solution covering the slice surface falls into the elution tank 2. Subsequently, the controller 9 controls the inlet valve 18, the drain valve 19, and the switching valve 40 to open, connecting the elution tank 2 to the pressure pump 17 and the storage tank 16. The storage tank 15 is connected to the pressure pump 17 and the storage tank 16 by means of a partition. The compartments separated by plate 37 contain cleaning solutions corresponding to different types of slides. Switching valve 40, under the control of controller 9, connects to the corresponding compartment according to the slide type. Pressurized pump 17, driven by replacement motor 21, pressurizes the cleaning solution stored in storage tank 15 and introduces it into elution tank 2, flushing away any residual eluent in elution tank 2. Then, only drain valve 19 is closed to disconnect elution tank 2 from storage tank 16, continuously injecting cleaning solution into elution tank 2. Once level sensor 20 detects that the cleaning solution level in elution tank 2 has reached the target, inlet valve 18 is closed. Disconnect the pressure pump 17 and the replacement motor 21. Then, control the elution motor 8 to connect to the rotating shaft 4. Under the power transmission of the elution motor 8, the rotating shaft 4 drives the elution clamp 3 to rotate, causing the sections to rotate in the cleaning solution. The rotation speed and time of the sections in the cleaning solution vary depending on the type of section. Frozen sections are rotated at a speed of 50-100 rpm for 5 minutes, and paraffin sections are rotated at a speed of 100-150 rpm for 5-10 minutes. After the initial cleaning is completed, the controller 9 controls the drain valve 19 to open, draining the cleaning solution in the elution tank 2 into the storage tank. In liquid tank 3 16, the elution motor 8 drives the rotating shaft 4 to restore the slide to its original position. The scanner 12 collects information on the slide surface. If there is residual elution solution on the slide surface, the controller 9 controls the pressurization pump 2 17, water inlet valve 18, and drain valve 19 to perform secondary cleaning to ensure that there is no residual elution solution on the slide surface and to avoid abnormal slide test results due to residual elution solution. During the staining process of frozen slides, the cooling unit 38 is connected to an external power source to maintain the temperature of the cleaning solution stored in liquid tank 2 15 at about 4°C to prevent tissue detachment.

[0025] Example 3: Please refer to Figure 1 , Figure 2 and Figure 4A rapid iterative TSA multiplex immunofluorescence staining kit includes an operating table 1. An elution tank 2 is installed on the upper side of the outer wall of the operating table 1. A spitting clip 3 is installed on the left side of the inner wall of the elution tank 2. The spitting clip 3 is connected to the elution tank 2 via a rotating shaft 4. A dropper 5 is installed on the rear side of the outer wall of the elution tank 2. The dropper 5 is connected to the operating table 1 via a moving component. A storage tank 6 is installed on the rear side of the inner wall of the operating table 1. A pressure pump 7 is installed on the upper side of the outer wall of the storage tank 6. The dropper 5 is connected to the pressure pump 7 via a connecting pipe. An elution motor 8 is installed on the left side of the outer wall of the pressure pump 7. The spitting clip 3, rotating shaft 4, moving component, and pressure pump 7 are respectively connected to a switch at the output end of the elution motor 8 via connecting shafts. The elution motor 8 is connected to a controller 9 installed on the front side of the outer wall of the operating table 1 via a signal line. A telescopic plate 22 is provided on the lower side of the outer wall of the detachment clamp 3, and a telescopic plate 23 is provided on the upper side of the outer wall of the liquid level sensor 20. Both the telescopic plate 22 and the telescopic plate 23 are fixedly connected to the rear side of the inner wall of the washing tank 2. Both the telescopic plate 22 and the telescopic plate 23 are provided with sealing rings 24 on their outer walls. A closed motor 25 is installed on the lower side of the outer wall of the washing tank 2. The telescopic plate 22 and the telescopic plate 23 are respectively connected to the switch at the output end of the closed motor 25 through connecting shafts. Both the telescopic plate 22 and the telescopic plate 23 are provided with leakage valves 26 through their surfaces. Both the closed motor 25 and the leakage valves 26 are connected to the controller 9 through signal lines. Furthermore, after the operator fixes the pretreated slides onto the elution clamp 3, and after the primary antibody is added, incubation is required before the secondary antibody can be added. After the operator adds the primary antibody to the slides, the controller 9 controls the switch at the output of the closed motor 25 to connect the telescopic plate 1 22 and the telescopic plate 23. Driven by the closed motor 25, the elution clamp 3 is isolated from the elution tank 2. The controller 9 controls the elution motor 8 to connect to the pressure pump 7 to pressurize the 37°C liquid stored in the storage tank 6 and allow it to flow out from the dropper 5. To avoid interference from the elution solution in the storage tank 6 with the multiple staining cycle, the structure of the storage tank 6 can be modified to that of the storage tank 2 15, allowing the pressure pump 7 to be freely switched between the compartments in the storage tank 6. The storage tank 6 contains liquid for incubation and insulation, such as water or washing solution. The 37°C liquid is added to the telescopic plate 22 and the secondary antibody 23. On the surface of the second shrink plate 23, the controller 9 controls the opening of the leakage valve 26 set on the surfaces of the first shrink plate 22 and the second shrink plate 23, allowing the 37°C liquid to flow downwards. Only a small amount of liquid is retained in the sealed space formed by the elution tank 2, the first shrink plate 22, and the second shrink plate 23 to maintain the incubation humidity and prevent the sections from drying out. A liquid-incubation space-liquid insulation structure is formed inside the elution tank 2. The sealing ring 24 set at the contact position between the first shrink plate 22 and the second shrink plate 23 and the elution tank 2 can prevent liquid leakage from the insulation structure. After the incubation is completed, the liquid in the elution tank 2 is drained, and the operator adds secondary antibody and continues incubation, maintaining the light-proof and constant temperature conditions during incubation to improve the staining stability of the sections. At the same time, the first shrink plate 22 and the second shrink plate 23 can isolate the sections from the external environment, preventing harmful substances from escaping and polluting the environment or the environment from interfering with the staining of the sections.

[0026] Example 4: Please refer to Figure 1 , Figure 3 , Figure 5 and Figure 7 A rapid iterative TSA multiplex immunofluorescence staining kit is provided, wherein a reagent box 27 is installed on the upper side of the outer wall of the second storage tank 15, a slide box 28 is installed on the upper side of the outer wall of the third storage tank 16, an opening and closing plate 29 is installed at the connection between the reagent box 27 and the slide box 28 and the operating table 1, a partition 30 is provided in the middle of the inner wall of the reagent box 27, a sliding groove 31 is provided on the lower side of the inner wall of the reagent box 27, a slider 32 is provided in the middle of the inner wall of the sliding groove 31, a push plate 33 is installed on the upper side of the outer wall of the slider 32, and the opening and closing plate 29 and the slider 32 are respectively connected to the switch at the output end of the closed motor 25 through a connecting shaft; A partition 37 is provided in the middle of the inner wall of the liquid storage tank 15. A refrigeration unit 38 is embedded in the inner walls of both the liquid storage tank 15 and the partition 37. A temperature sensor 39 is installed on the lower side of the inner wall of the liquid storage tank 15. A switching valve 40 is installed at the connection between the liquid storage tank 15 and the pressurizing pump 17. The refrigeration unit 38, the temperature sensor 39 and the switching valve 40 are all connected to the controller 9 through signal lines. The reagent box 27 and the slide box 28 are equipped with heat-insulating jacket 41. A circulation pump 42 is installed on the upper side of the outer wall of the pressure pump 2 17. The heat-insulating jacket 41 is connected to the circulation pump 42 through connecting pipes. The circulation pump 42 is connected to the switch at the output end of the replacement motor 21 through a connecting shaft. Furthermore, paraffin sections can be stained after dewaxing, hydration, antigen retrieval, and endogenous enzyme blocking treatment. Frozen sections can be stained after fixation, permeabilization, and anti-detachment treatment. To avoid operator errors during multiple staining processes and confusion in the order of primary and secondary antibody addition, in the initial stage of multiple staining cycles, the controller 9 controls the switch at the output of the closed motor 25 to connect to the opening and closing plate 29 of the slide box 28, opening the slide box 28. After the operator fixes the pre-treated slides stored in the slide box 28 onto the detachment clip 3, the slide box 28 is closed, and then the closed motor 25 is connected. 5. Open reagent box 27. The primary and secondary antibodies stored in reagent box 27 are located at a certain distance from the front end of operating table 1. Controller 9 controls the slider 32, located below the push plate 33 for placing the primary and secondary antibodies, connected to the closed motor 25, according to the operator's staining sequence. Driven by the closed motor 25, the slider 32 slides within the chute 31, moving the push plate 33 for placing the primary and secondary antibodies to the front end of operating table 1 sequentially according to the operator's staining sequence. After the primary antibody is added, the push plate 33 is reset, and reagent box 27 is closed via the opening / closing plate 29. After the primary antibody is added... After processing, open reagent box 27, and move the push plate 33 containing the secondary antibody to the front of operating table 1 using slider 32. After adding the secondary antibody, reset push plate 33 and close reagent box 27. Repeat the operation to complete multiple staining cycles. During continuous staining, to ensure the reagents and slides in reagent box 27 and slide box 28 are well preserved, controller 9 controls the switch at the output of replacement motor 21 to connect to circulation pump 42. Replacement motor 21 continuously supplies power to circulation pump 42, and the temperature of the circulating liquid stored in storage tank 15 is reduced by connecting cooling unit 38 and external power supply. Sensor 2 39 stops cooling when the temperature inside the storage tank 2 15 drops to 4-8℃. The low-temperature circulating liquid is then transported to the insulation jacket 41 by the circulation pump 42 to maintain the temperature inside the reagent box 27 and the slide box 28. Depending on the type of reagent required for staining, the number of partitions 30 in the reagent box 27 can be changed. For example, if only primary and secondary antibodies are needed for staining, only one partition 30 is set in the reagent box 27. If multiple protein antigens on the slides need to be treated, multiple partitions 30 can be set in the reagent box 27 to separate the different antibodies and transport them sequentially to the front end of the operating table 1 for the operator to use.

[0027] Example 5: Please refer to Figure 1 , Figure 2 , Figure 3 and Figure 4A rapid iterative TSA multiplex immunofluorescence staining kit is described. The operating table 1 has a storage tank 2 15 and a storage tank 3 16 installed on the left and right sides of its inner wall, respectively. A pressure pump 2 17 is installed on the outer wall of storage tank 2 near the elution tank 2. The pressure pump 2 17 is connected to an inlet valve 18 installed on the left side of the inner wall of the elution tank 2 via a connecting pipe. Storage tank 2 15 is connected to a drain valve 19 installed on the lower side of the inner wall of the elution tank 2 via a connecting pipe. A level sensor 20 is embedded on the upper side of the outer wall of the elution clamp 3, close to the inner wall of the elution tank 2. A replacement motor 21 is installed on the lower side of the outer wall of pressure pump 2 17. Pressure pump 2 17 is connected to a switch at the output end of the replacement motor 21 via a connecting shaft. The inlet valve 18, drain valve 19, level sensor 20, and replacement motor 21 are all connected to a controller 9 via signal lines. A telescopic plate 22 is provided on the lower side of the outer wall of the detachment clamp 3, and a telescopic plate 23 is provided on the upper side of the outer wall of the liquid level sensor 20. Both the telescopic plate 22 and the telescopic plate 23 are fixedly connected to the rear side of the inner wall of the washing tank 2. Both the telescopic plate 22 and the telescopic plate 23 are provided with sealing rings 24 on their outer walls. A closed motor 25 is installed on the lower side of the outer wall of the washing tank 2. The telescopic plate 22 and the telescopic plate 23 are respectively connected to the switch at the output end of the closed motor 25 through connecting shafts. Both the telescopic plate 22 and the telescopic plate 23 are provided with leakage valves 26 through their surfaces. Both the closed motor 25 and the leakage valves 26 are connected to the controller 9 through signal lines. Furthermore, during multiple staining cycles, the slides need to be washed after the primary antibody addition and before the secondary antibody addition to remove unbound primary antibody residue. The primary antibody incubation solution often contains serum proteins or blocking agents; incomplete washing can hinder the binding efficiency of the secondary and primary antibodies. After the primary antibody addition and incubation, the controller 9 controls the replacement motor 21 to drive the pressure pump 17, pressurizing the washing solution stored in the storage tank 15 and introducing it into the elution tank 2 through the inlet valve 18. To prevent slide detachment or dissociation due to a sudden temperature change from a 37°C environment to a 4°C environment, the washing solution is washed... After the incubation solution below the first telescopic plate 22 in the elution tank 2 is discharged through the drain valve 19, the water inlet valve 18 is set at a position lower than the corresponding height of the first telescopic plate 22. When the cleaning solution is added to the elution tank 2 through the water inlet valve 18, the leakage valves 26 on the first telescopic plate 22 and the second telescopic plate 23 are opened simultaneously. This allows the incubation solution, which is at a different temperature from the cleaning solution, to mix with the cleaning solution and neutralize the temperature. Subsequently, the first telescopic plate 22 and the second telescopic plate 23 are contracted under the drive of the closed motor 25, allowing the slides to come into contact with the cleaning solution. This prevents the slides from directly entering the cleaning environment from the incubation environment, which could cause tissue detachment or dissociation due to temperature differences.

[0028] Working principle: The controller 9 controls the switch at the output of the closed motor 25 to connect to the opening and closing plate 29 of the slide box 28, opening the slide box 28. The operator places the pre-processed slides in front of the elution clamp 3. The controller 9 controls the switch at the output of the elution motor 8 to connect to the elution clamp 3. Driven by the elution motor 8, the elution clamp 3 holds and fixes the slides. The opening and closing plate 29 closes the slide box 28. Then, the closed motor 25 is connected to the reagent box 27, opening the reagent box 27. The primary and secondary antibodies stored in the reagent box 27 are located at a certain distance from the front of the operating table 1. According to the operator's staining process, the controller 9 controls the slider 32 located below the push plate 33 for placing the primary and secondary antibodies to connect to the closed motor 25. Driven by the closed motor 25, the slider 32 slides in the groove 31, moving the push plate 33 for placing the primary antibody to the front of the operating table 1. After the primary antibody is added, the push plate 33 for placing the primary antibody is reset, and the opening and closing plate 29 closes the reagent box 27. When closed, controller 9 controls the switch at the output of the closed motor 25 to connect the telescopic plate 1 22 and the telescopic plate 23. Driven by the closed motor 25, the detachment clamp 3 is isolated from the washing tank 2. Controller 9 controls the washing motor 8 to connect to the pressurization pump 7 to pressurize the 37°C liquid stored in the storage tank 6 and allow it to flow out from the dripper 5. The 37°C liquid drips onto the surface of the telescopic plate 23. Controller 9 controls the leakage valve 26 installed on the surfaces of the telescopic plates 1 22 and 23 to open, allowing... The 37°C liquid flows downwards, and only a small amount of liquid is retained in the closed space formed by the elution tank 2, the first telescopic plate 22, and the second telescopic plate 23 to maintain the humid state of incubation. A liquid-incubation space-liquid insulation structure is formed inside the elution tank 2. The sealing rings 24 set at the contact positions of the first telescopic plate 22 and the second telescopic plate 23 with the elution tank 2 can prevent liquid leakage from the insulation structure. After the incubation is completed, the drain valve 19 and the leakage valve 26 are opened to drain the liquid in the elution tank 2. After the primary antibody is added, the controller 9 opens the inlet valve 18, drain valve 19, and switching valve 40, connecting the elution tank 2 to the pressure pump 17 and the storage tank 16. Under the control of the controller 9, the switching valve 40 connects to the corresponding cleaning solution in the storage tank 15 according to the type of slice. Driven by the replacement motor 21, the pressure pump 17 pressurizes the cleaning solution in the storage tank 15 and introduces it into the elution tank 2 to clean it. Then, only the drain valve 19 is closed to disconnect the elution tank 2 from the storage tank 16, allowing the cleaning solution to be continuously injected into the elution tank 2. The cleaning solution is then injected into the elution tank 2 until the level sensor 20 detects that the height of the cleaning solution in the elution tank 2 has reached a certain level. After labeling, the inlet valve 18 is closed, the connection between the pressure pump 17 and the replacement motor 21 is disconnected, and then the elution motor 8 is connected to the rotating shaft 4. Under the power transmission of the elution motor 8, the rotating shaft 4 drives the elution clamp 3 to rotate, so that the slide rotates in the cleaning solution. The rotation speed of the rotating shaft 4 is adjusted according to the different types of slides to complete the cleaning. The primary antibody addition and incubation operations are repeated. The push plate 33 for placing the secondary antibody is moved to the front end of the operating table 1 by the slider 32. After the secondary antibody addition is completed, the slider 32 resets the push plate 33 and closes the reagent box 27 by the opening and closing plate 29. Incubation is carried out by constructing a liquid-incubation space-liquid insulation structure. After staining is completed, the heating wire 13 of the controller 9 is connected to an external power source to heat the eluent stored in the storage tank 6. Heating stops when the temperature sensor 14 inside the storage tank 6 detects that the eluent temperature has risen to 37°C. The controller 9 controls the switch at the output of the elution motor 8 to connect to the pressurization pump 7. Driven by the elution motor 8, the 37°C eluent stored in the storage tank 6 is pressurized and delivered to the storage chamber 36 of the dropper 5 for temporary storage. Subsequently, the controller 9 controls the regulating valve 35 to adjust the flow rate at the droplet port 34 and controls the elution motor 8 to synchronously deliver power to the moving component to adjust the position of the dropper 5. The switch at the output of the elution motor 8 is connected to the telescopic shaft 10 and the rotating shaft 11. Driven by the elution motor 8, the telescopic shaft 10 adjusts the height of the dropper 5, causing the eluent to drop from different heights. The eluent is added by rotating shaft 2 11 and adjusting the angle of the dropper 5 to ensure that the eluent is evenly dripped onto the slice surface. After the eluent is added, the slice is scanned by scanner 12 to obtain the distribution of the eluent on the slice surface. If the eluent does not completely cover the slice surface due to tissue adsorption, controller 9 controls elution motor 8 to send power to rotating shaft 2 11. Driven by elution motor 8, rotating shaft 2 11 drives the elution clamp 3 to adjust the angle of the slice. When the weight sensor 43 detects that the weight of the slice held by the elution clamp 3 has increased to the set value, the dropper 5 stops adding eluent even if the eluent coverage on the slice surface is uneven. The angle of the elution clamp 3 is adjusted by rotating shaft 1 4 to make the eluent evenly cover the slice. At the same time, infrared sensor 44 collects information on the thickness of the eluent covering the slice surface to ensure that the eluent coverage thickness meets the requirements. The controller 9 records the time when the elution solution covers the slide surface. After the set time is reached according to the slide type, the controller 9 controls the elution motor 8 to drive the rotating shaft 4 to flip the elution clamp 3, causing the slide to flip 180°. The elution solution covering the slide surface falls into the elution tank 2. The cleaning operation after the primary antibody is added is repeated. The elution motor 8 drives the rotating shaft 4 to return the slide to its original position. The scanner 12 collects the information of the slide surface. If there is elution solution residue on the slide surface, the controller 9 controls the pressure pump 17, water inlet valve 18 and drain valve 19 to perform a secondary cleaning until there is no elution solution residue on the slide surface. The operation is repeated for multiple staining cycles.

[0029] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A rapid iterative TSA multiplex immunofluorescence staining kit, characterized in that: The system includes an operating table (1), an elution tank (2) installed on the upper side of the outer wall of the operating table (1), a detachment clamp (3) installed on the left side of the inner wall of the elution tank (2), the detachment clamp (3) being connected to the elution tank (2) via a rotating shaft (4), a dropper (5) installed on the rear side of the outer wall of the elution tank (2), the dropper (5) being connected to the operating table (1) via a moving component, and a storage tank (6) installed on the rear side of the inner wall of the operating table (1). 6) A pressure pump (7) is installed on the upper side of the outer wall. The drip feeder (5) is connected to the pressure pump (7) through a connecting pipe. A washing motor (8) is installed on the left side of the outer wall of the pressure pump (7). The detachment clamp (3), the rotating shaft (4), the moving component and the pressure pump (7) are respectively connected to the switch at the output end of the washing motor (8) through the connecting shaft. The washing motor (8) is connected to the controller (9) installed on the front side of the outer wall of the operating table (1) through a signal line.

2. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 1, characterized in that: The moving assembly consists of a telescopic shaft (10) and a rotating shaft (11). The telescopic shaft (10) is installed on the rear side of the outer wall of the elution tank (2), and the rotating shaft (11) is installed on the front side of the outer wall of the telescopic shaft (10). The telescopic shaft (10) and the rotating shaft (11) are respectively connected to the elution motor (8) through connecting shafts. A scanner (12) is installed on the front side of the outer wall of the moving assembly. The scanner (12) is connected to the controller (9) through a signal line. Heating wires (13) are installed on both the front and rear sides of the inner wall of the first storage tank (6). A temperature sensor (14) is installed on the rear side of the inner wall of the first storage tank (6). The heating wires (13) are connected to the temperature sensor (14) through a signal line.

3. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 1, characterized in that: The operating table (1) has a storage tank 2 (15) and a storage tank 3 (16) installed on the left and right sides of the inner wall. The outer wall of the storage tank 2 (15) is close to the elution tank (2) and a pressure pump 2 (17) is installed. The pressure pump 2 (17) is connected to the water inlet valve (18) installed on the left side of the inner wall of the elution tank (2) through a connecting pipe. The storage tank 2 (15) is connected to the drain valve (19) installed on the lower side of the inner wall of the elution tank (2) through a connecting pipe. The upper side of the outer wall of the elution clamp (3) is close to the inner wall of the elution tank (2) and a liquid level sensor (20) is embedded. The lower side of the outer wall of the pressure pump 2 (17) is equipped with a replacement motor (21). The pressure pump 2 (17) is connected to the switch at the output end of the replacement motor (21) through a connecting shaft. The water inlet valve (18), the drain valve (19), the liquid level sensor (20) and the replacement motor (21) are all connected to the controller (9) through signal lines.

4. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 3, characterized in that: The lower side of the outer wall of the detachment clamp (3) is provided with a telescopic plate 1 (22), and the upper side of the outer wall of the liquid level sensor (20) is provided with a telescopic plate 2 (23). Both the telescopic plate 1 (22) and the telescopic plate 2 (23) are fixedly connected to the rear side of the inner wall of the washing tank (2). Both the outer walls of the telescopic plate 1 (22) and the telescopic plate 2 (23) are provided with sealing rings (24). A closed motor (25) is installed on the lower side of the outer wall of the washing tank (2). The telescopic plate 1 (22) and the telescopic plate 2 (23) are respectively connected to the switch at the output end of the closed motor (25) through the connecting shaft. Both the surface of the telescopic plate 1 (22) and the telescopic plate 2 (23) are provided with a leakage valve (26). Both the closed motor (25) and the leakage valve (26) are connected to the controller (9) through the signal line.

5. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 4, characterized in that: A reagent box (27) is installed on the upper side of the outer wall of the second liquid storage tank (15), and a slice box (28) is installed on the upper side of the outer wall of the third liquid storage tank (16). A hinged plate (29) is installed at the connection between the reagent box (27) and the slice box (28) and the operating table (1). A partition plate (30) is provided in the middle of the inner wall of the reagent box (27). A slide groove (31) is provided on the lower side of the inner wall of the reagent box (27). A slider (32) is provided in the middle of the inner wall of the slide groove (31). A push plate (33) is installed on the upper side of the outer wall of the slider (32). The hinged plate (29) and the slider (32) are respectively connected to the switch at the output end of the closed motor (25) through the connecting shaft.

6. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 2, characterized in that: The dripper (5) consists of a drip inlet (34), a regulating valve (35), and a liquid storage chamber (36). The liquid storage chamber (36) is installed on the lower side of the outer wall of the rotating shaft (11). The drip inlet (34) is installed on the lower side of the outer wall of the liquid storage chamber (36). The regulating valve (35) is installed at the connection between the drip inlet (34) and the liquid storage chamber (36). The liquid storage chamber (36) is connected to the pressure pump (7) through a connecting pipe. The regulating valve (35) is connected to the controller (9) through a signal line.

7. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 5, characterized in that: A partition plate (37) is provided in the middle of the inner wall of the liquid storage tank (15). A refrigeration unit (38) is embedded in the inner wall of both the liquid storage tank (15) and the partition plate (37). A temperature sensor (39) is installed on the lower side of the inner wall of the liquid storage tank (15). A switching valve (40) is installed at the connection between the liquid storage tank (15) and the pressure pump (17). The refrigeration unit (38), the temperature sensor (39) and the switching valve (40) are all connected to the controller (9) through signal lines.

8. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 7, characterized in that: The reagent box (27) and the slide box (28) are equipped with a heat-insulating jacket (41). A circulation pump (42) is installed on the upper side of the outer wall of the pressure pump (17). The heat-insulating jacket (41) is connected to the circulation pump (42) through a connecting pipe. The circulation pump (42) is connected to the switch at the output end of the replacement motor (21) through a connecting shaft.

9. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 1, characterized in that: A weight sensor (43) is installed at the connection between the detachment clamp (3) and the rotating shaft (4). The weight sensor (43) is connected to the controller (9) via a signal line.

10. The rapid iterative TSA multiplex immunofluorescence staining kit according to claim 1, characterized in that: An infrared sensor (44) is embedded on the left side of the inner wall of the washing tank (2) at the same height as the elution clamp (3). The infrared sensor (44) is connected to the controller (9) through a signal line.

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