Device for cyclically dyeing cells or tissues and use method

Abstract

The invention discloses a device for cyclic staining of cells or tissues and a use method. The device comprises an upper cover, a bottom cover, a section, a liquid inlet, a liquid outlet, a positive electrode and a negative electrode, wherein the upper cover, the bottom cover and the cutting sheet are pressed together in sequence from top to bottom, the liquid inlet and the liquid outlet are respectively arranged on the left side and the right side of the upper cover, the positive electrode is arranged in the liquid inlet, and the negative electrode is arranged in the liquid outlet. According to the device and the using method, fluorescent staining can be conveniently carried out on cells or tissues, and staining fluorescent signals on the surfaces of the tissue cells can be rapidly and nondestructively removed.

Description

Technical Field

[0001] The present invention relates to the technical field of medical testing and analysis, and in particular to a device for cyclic staining of cells or tissues and a method for using the device. Background Art

[0002] Because tumors are constantly evolving and heterogeneous, the tumor immune microenvironment interacts with tumor cells. Numerous clinical cases and scientific research indicate that in-depth analysis of the tumor immune microenvironment will be key to overcoming barriers to immunotherapy. Analysis of the immune microenvironment, or immune infiltration, essentially aims to clearly characterize the phenotype, abundance, and in situ spatial distribution of immune cells within tumor tissue.

[0003] Immunohistochemistry / immunofluorescence imaging technology is a technology that applies the basic principle of immunology - antigen-antibody reaction, to qualitatively and positionally analyze antigens or antibody substances in tissues or cells. It is a common means of describing the spatial omics information of the tumor microenvironment and is widely used in clinical pathological diagnosis. However, traditional immunohistochemistry / fluorescence staining can only mark 1-3 colors on a tissue section, and cannot mark a large number of target molecules, and it is difficult to handle subtle pathological tissues that require multiple staining. Although staining multiple sections continuously can increase the number of detection markers, the spatial misalignment between sections can easily lead to missed detections, and it is impossible to accurately depict the spatial position relationship of various cells in the microenvironment and waste rare sections.

[0004] Cyclic fluorescence imaging is a common strategy used in multi-label immunofluorescence imaging to increase analytical throughput. It constructs high-dimensional images by performing multiple rounds of immunofluorescence panoramic scanning on conventional sections. Cyclic imaging techniques that use chemical reagents to remove fluorescent signals carry the risk of damaging cell surface antigens during the signal removal process, resulting in reduced accuracy after multiple cycles. The relatively mild antibody elution method, which removes fluorescent signals by disrupting the antigen-antibody interaction through competitive binding, is not only time-consuming but also prone to residual signal, affecting analytical accuracy.

[0005] To this end, this patent application proposes a method for using an electric field to generate a controllable dose of a fluorescence quenching chemical component in situ. This chemical component can rapidly quench the fluorescence signal of dyes present in tissues or cells. This principle is further exploited to construct a microfluidic device chip, integrating the dyeing and decolorization processes into a single device. Combined with a fluid control system, this device can enable cyclic imaging analysis of cells or tissues. Summary of the Invention

[0006] In view of this, the present invention provides a device and a method for use for cyclic staining of cells or tissues.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A device for cyclic staining of cells or tissues, comprising an upper cover, a bottom cover, a slice, a liquid inlet, a liquid outlet, a positive electrode, and a negative electrode; wherein the upper cover, the bottom cover, and the slice are pressed together in order from top to bottom, the liquid inlet and the liquid outlet are respectively arranged on the left and right sides of the upper cover, the positive electrode is placed in the liquid inlet, and the negative electrode is placed in the liquid outlet.

[0009] Preferably, the slice is set to be square, and a hollow square area is provided on the bottom cover, and the slice can be fixed in the hollow square area on the bottom cover.

[0010] Preferably, the bottom cover and the upper cover can be sealed together.

[0011] Preferably, both the upper cover and the bottom cover are provided with adhesive areas, which can be used to adhere the entire device.

[0012] Preferably, the bonding area is sealed with a double-sided water-resistant solid tape.

[0013] Preferably, the device further comprises a circulation channel, and the thickness of the circulation channel must be in the range of 50-150 microns.

[0014] Preferably, in order to facilitate the operation of the entire experimental process, the liquid inlet and the liquid outlet can also be placed externally, that is, the liquid outlet and the liquid inlet are respectively located on both sides of the positive and negative electrode insertion ports, ensuring that the liquid added to the liquid inlet can flow through the positive electrode area, pushing the liquid in this area to flow through the slice analysis area.

[0015] A device and method for cyclic staining of cells or tissues, comprising the following steps:

[0016] Step 1: Pre-treatment of paraffin sections for fluorescent immunohistochemical staining;

[0017] Step 2: Encapsulate the tissue slice or cell slide with the chip;

[0018] Step 3: (1) Wash three times with PBST (1X) buffer, 3 min each time;

[0019] (2) After incubation with endogenous catalase removal solution at room temperature for 10 min, the cells were washed three times with PBST immersion and shaking;

[0020] Step 4: (1) Add an appropriate amount of immune blocking solution from the chip inlet for blocking. Incubate at room temperature for 10 minutes, and then discard the blocking solution in the chip.

[0021] (2) An appropriate amount of primary antibody solution was loaded into the chip from the liquid inlet, incubated at 4°C overnight for 16 h, equilibrated at room temperature for 45 min, and washed three times with PBST and oscillation for 3 min each time;

[0022] (3) Remove the PBST in the chip and load an appropriate amount of pika and rabbit universal enzyme-labeled secondary antibody solution into the chip from the liquid inlet. Incubate at room temperature for 15 min, then add PBST and wash with shaking three times, each time for 3 min.

[0023] (4) After removing the PBST in the chip, add an appropriate amount of fluorescent dye working solution into the chip from the liquid inlet and incubate at room temperature for 10 minutes;

[0024] (5) Add PBS to the liquid inlet and wash with shaking for 3 times, each time for 5 minutes;

[0025] Step 5: After the chip is filled with PBS solution, imaging is performed;

[0026] Step 6: Place positive and negative electrodes at the inlet and outlet, respectively, and energize the electrodes with a voltage of 3-16 volts for 1-5 minutes to ensure that sufficient active ingredients are generated near the positive electrode in the solution.

[0027] Step 7: (1) Turn off the power to the electrode, add PBST solution to the liquid inlet to push the solution containing the active ingredient around the electrode and flow through the tissue slice area of ​​the chip, and then suck out the excess solution at the liquid outlet for 30-150 seconds;

[0028] (2) Add PBST to the liquid inlet for washing three times, and finally fill it with PBST solution;

[0029] Step 8: (1) After discarding the PBST solution in the chip, load an appropriate amount of elution solution preheated at 37°C into the liquid inlet to cover the tissue section area and leave it at room temperature for 3-5 minutes;

[0030] (2) After discarding the eluent, reload an appropriate amount of elution solution preheated at 37°C from the liquid inlet, place the chip in a wet box and incubate at 37°C for 25-30 minutes. After incubation, wash with PBST three times, each time for 3 minutes.

[0031] Step 9: Repeat steps 4-5 for multiple rounds of labeling, staining, and imaging.

[0032] Preferably, the step 1 comprises the following steps:

[0033] (1) Dewaxing and hydration

[0034] ① Dewax the sections in xylene twice, 10 min each time;

[0035] ② Then add anhydrous ethanol, anhydrous ethanol, 95% ethanol, and 85% ethanol in sequence and soak at room temperature for 5 minutes each time;

[0036] ③ Rinse the slices twice with tap water to wash off the ethanol;

[0037] (2) Antigen retrieval

[0038] Select a repair solution suitable for the selected primary antibody to repair the tissue. First, boil it in a microwave at high temperature for 5 minutes, then immerse the slices in the repair solution and repair it at medium temperature in the microwave for 15 minutes, and cool it at room temperature for more than 2 hours.

[0039] Preferably, the specific operation of step 3 (2) is: adding an appropriate amount of PBST to the liquid inlet, letting it stand for 1-3 minutes, and sucking out the solution at the liquid outlet; repeating the above operation 3 times.

[0040] Compared with the prior art, the present invention has achieved the following technical effects:

[0041] The device and method of the present invention can conveniently perform fluorescent staining on cells or tissues, and quickly and non-destructively remove the stained fluorescent signals on the surface of tissue cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic structural diagram of a device for cyclic staining of cells or tissues and a method of use of the present invention;

[0043] Figure 2 A diagram of a real device for a device for cyclic staining of cells or tissues and a method of use of the present invention;

[0044] Figure 3 A diagram illustrating the bonding of an upper cover and a lower cover of a device for cyclic staining of cells or tissues and a method of use of the present invention;

[0045] Figure 4 and Figure 5 A diagram of a cell or tissue circulation staining device and a method for using the device for cell or tissue circulation staining according to the present invention;

[0046] Figure 6 A flow chart of a device for cyclic staining of cells or tissues and a method of use of the present invention;

[0047] Figure 7 A diagram showing the results of cell staining and decolorization analysis of a device and method for cyclic staining of cells or tissues according to the present invention;

[0048] Figure 8 A comparison diagram of the fluorescence images of 20 rounds of cyclic staining with different antibodies and the fluorescence image of the first staining in the device and method for cyclic staining of cells or tissues of the present invention;

[0049] Figure 9This is a comparison diagram of the fluorescence image of a tissue section after six decolorization cycles and the effect of the initial staining according to the device and method for cyclic staining of cells or tissues of the present invention. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] Example 1:

[0052] The present invention discloses a device for cyclic staining of cells or tissues, comprising an upper cover 1, a bottom cover 2, a slice 3, a liquid inlet 4, a liquid outlet 5, a positive electrode 6, and a negative electrode 7. The upper cover 1, the bottom cover 2, and the slice 3 are pressed together in order from top to bottom, the liquid inlet 4 and the liquid outlet 5 are respectively arranged on the left and right sides of the upper cover 1, the positive electrode 6 is placed in the liquid inlet 4, and the negative electrode 7 is placed in the liquid outlet 5. The slice 3 is configured as a square, and the bottom cover 2 is provided with a hollow square area, and the slice 3 can be fixed to the hollow square area on the bottom cover 2. The bottom cover 2 and the upper cover 1 can be sealed together. The upper cover 1 and the bottom cover 2 are both provided with an adhesive area 8, which can be used to bond the entire device. The adhesive area 8 is sealed with a double-sided water-resistant solid tape. The device also includes a circulation pipe 9, and the thickness of the circulation pipe 9 must range from 50 to 150 microns.

[0053] In order to facilitate the operation of the entire experimental process, the liquid inlet 4 and the liquid outlet 5 can also be placed externally, that is, the liquid outlet 5 and the liquid inlet 4 are located on both sides of the positive and negative electrode insertion ports, respectively, to ensure that the liquid added to the liquid inlet 4 can flow through the positive electrode 6 area, pushing the liquid in this area to flow through the slice analysis area. Figure 1 , the actual device diagram is as follows Figure 2 , the bonding diagram of the upper cover and the lower cover is as follows Figure 3 .

[0054] Example 2:

[0055] The present invention can also reserve liquid inlet and outlet holes and electrode holes on the upper cover of the device, such as Figure 4 As shown, a sealing rubber ring is fixed to the bottom layer of the upper cover to seal against the glass slide. The thickness of the sealing rubber ring is 100 microns. The inner diameter of the sealing rubber ring is longer than the outer edge of the inlet and outlet holes, and the width of the sealing rubber ring is wider than the edge of the tissue section or cell disc area.

[0056] Example 3:

[0057] The present invention also includes a design that does not use an external platinum wire electrode, but instead uses a design that fixes the electrode and the chip together, such as Figure 5 As shown, its structural feature is that the electrodes are pre-fixed to the chip below the inlet and outlet ports. The fixing technology can be spray coating or chemical etching. After the upper and lower layers of the chip are closed, the exposed ends of the fixed electrodes can be used to connect to an external circuit. The electrodes used in the device include but are not limited to platinum electrodes, gold electrodes, and graphite electrodes.

[0058] Example 4:

[0059] like Figure 6 As shown, a device and method for circulating staining of cells or tissues include the following steps:

[0060] Step 1: Pre-treatment of paraffin sections for fluorescent immunohistochemical staining;

[0061] (1) Dewaxing and hydration

[0062] ① Dewax the sections in xylene twice, 10 min each time;

[0063] ② Then add anhydrous ethanol, anhydrous ethanol, 95% ethanol, and 85% ethanol in sequence and soak at room temperature for 5 minutes each time;

[0064] ③ Rinse the slices twice with tap water to wash off the ethanol;

[0065] (2) Antigen retrieval

[0066] Select a repair solution suitable for the selected primary antibody to repair the tissue. First, boil it in a microwave at high temperature for 5 minutes, then immerse the slices in the repair solution and repair it at medium temperature in the microwave for 15 minutes, and cool it at room temperature for more than 2 hours.

[0067] Step 2: Encapsulate the tissue slice or cell slide with the chip;

[0068] Step 3: (1) Wash three times with PBST (1X) buffer, 3 min each time;

[0069] (2) Then, incubate with endogenous catalase removal solution at room temperature for 10 minutes, and then wash three times with PBST immersion and shaking. The specific operation is as follows: add an appropriate amount of PBST to the liquid inlet, let it stand for 1-3 minutes, and then aspirate the solution at the liquid outlet. Repeat the above operation three times.

[0070] Step 4: (1) Add an appropriate amount of immune blocking solution from the chip inlet for blocking. Incubate at room temperature for 10 minutes, and then discard the blocking solution in the chip.

[0071] (2) An appropriate amount of primary antibody solution was loaded into the chip from the liquid inlet, incubated at 4°C overnight for 16 h, equilibrated at room temperature for 45 min, and washed three times with PBST and oscillation for 3 min each time;

[0072] (3) Remove the PBST in the chip and load an appropriate amount of pika and rabbit universal enzyme-labeled secondary antibody solution into the chip from the liquid inlet. Incubate at room temperature for 15 min, then add PBST and wash with shaking three times, each time for 3 min.

[0073] (4) After removing the PBST in the chip, add an appropriate amount of fluorescent dye working solution into the chip from the liquid inlet and incubate at room temperature for 10 minutes;

[0074] (5) Add PBS to the liquid inlet and wash with shaking for 3 times, each time for 5 minutes;

[0075] Step 5: After the chip is filled with PBS solution, imaging is performed;

[0076] Step 6: Place positive and negative electrodes at the inlet and outlet, respectively, and energize the electrodes with a voltage of 3-16 volts for 1-5 minutes to ensure that sufficient active ingredients are generated near the positive electrode in the solution.

[0077] Step 7: (1) Turn off the power to the electrode, add PBST solution to the liquid inlet to push the solution containing the active ingredient around the electrode and flow through the tissue slice area of ​​the chip, and then suck out the excess solution at the liquid outlet for 30-150 seconds;

[0078] (2) Add PBST to the liquid inlet for washing three times, and finally fill it with PBST solution;

[0079] Step 8: (1) After discarding the PBST solution in the chip, load an appropriate amount of elution solution preheated at 37°C into the liquid inlet to cover the tissue section area and leave it at room temperature for 3-5 minutes;

[0080] (2) After discarding the eluent, reload an appropriate amount of elution solution preheated at 37°C from the liquid inlet, place the chip in a wet box and incubate at 37°C for 25-30 minutes. After incubation, wash with PBST three times, each time for 3 minutes.

[0081] Step 9: Repeat steps 4-5 for multiple rounds of labeling, staining, and imaging.

[0082] Example 5:

[0083] like Figure 7 The fluorescence images and intensities shown demonstrate that the electrochemically assisted staining method can be applied to a variety of fluorescent materials. PE (encapsulated within the protein shell) also exhibits a strong quenching effect. This strategy, regardless of the location of the fluorescent molecules, can remove over 95% of the signal from intracellular immunofluorescence probes, demonstrating the versatility of this device.

[0084] like Figure 8 The fluorescence images of cells after 20 decolorization cycles within the device are compared with the initial staining results. The fluorescence intensity clearly shows that after 20 cycles, the cells can still be well immunofluorescently stained. This indicates that the device of the present invention can perform at least 20 cycles of staining.

[0085] Example 6:

[0086] like Figure 9 As shown, the fluorescence images of the tissue sections after 6 decolorization cycles are compared with the effects of the first staining. It can be clearly seen from the fluorescence intensity that after 6 decolorization cycles, the tissue sections can still be well stained with fluorescent immunohistochemistry of Ki67 antigen.

[0087] The above description is merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A device for cyclic staining of cells or tissues, characterized in that The invention comprises an upper cover (1), a bottom cover (2), a slice (3), a liquid inlet (4), a liquid outlet (5), a positive electrode (6) and a negative electrode (7); wherein the upper cover (1), the bottom cover (2) and the slice (3) are pressed together in order from top to bottom, the liquid inlet (4) and the liquid outlet (5) are respectively arranged on the left and right sides of the upper cover (1), the positive electrode (6) is placed in the liquid inlet (4), and the negative electrode (7) is placed in the liquid outlet (5).

2. The device for cyclic staining of cells or tissues according to claim 1, characterized in that: The slice (3) is configured as a square, a hollow square area is provided on the bottom cover (2), and the slice (3) can be fixed to the hollow square area on the bottom cover (2).

3. The device for cyclic staining of cells or tissues according to claim 1, characterized in that: The bottom cover (2) and the upper cover (1) can be sealed together.

4. The device for cyclic staining of cells or tissues according to claim 1, characterized in that: The upper cover (1) and the bottom cover (2) are both provided with an adhesive area (8) which can be used to adhere the entire device.

5. The device for cyclic staining of cells or tissues according to claim 4, characterized in that: The adhesive area (8) is sealed with a double-sided water-resistant solid tape.

6. The device for cyclic staining of cells or tissues according to claim 1, characterized in that: The device also includes a circulation channel (9), and the thickness of the circulation channel (9) must be in the range of 50-150 microns.

7. The device for cyclic staining of cells or tissues according to claim 1, characterized in that: In order to facilitate the operation of the entire experimental process, the liquid inlet (4) and the liquid outlet (5) can also be externally located, that is, the liquid outlet (5) and the liquid inlet (4) are respectively located on both sides of the positive and negative electrode insertion ports, ensuring that the liquid added to the liquid inlet (4) can flow through the positive electrode (6) area, pushing the liquid in this area to flow through the slice analysis area.

8. A device and method for cyclic staining of cells or tissues, characterized in that: The following steps are involved: Step 1: Pre-treatment of paraffin sections for fluorescent immunohistochemical staining; Step 2: Encapsulate the tissue slice or cell slide with the chip; Step 3: (1) Wash three times with PBST (1X) buffer, 3 min each time; (2) After incubation with endogenous catalase removal solution at room temperature for 10 min, the cells were washed three times with PBST immersion and shaking; Step 4: (1) Add an appropriate amount of immune blocking solution from the chip inlet for blocking. Incubate at room temperature for 10 minutes, and then discard the blocking solution in the chip. (2) An appropriate amount of primary antibody solution was loaded into the chip from the liquid inlet, incubated at 4°C overnight for 16 h, equilibrated at room temperature for 45 min, and washed three times with PBST and oscillation for 3 min each time; (3) Remove the PBST in the chip and load an appropriate amount of pika and rabbit universal enzyme-labeled secondary antibody solution into the chip from the liquid inlet. Incubate at room temperature for 15 min, then add PBST and wash with shaking three times, each time for 3 min. (4) After removing the PBST in the chip, add an appropriate amount of fluorescent dye working solution into the chip from the liquid inlet and incubate at room temperature for 10 minutes; (5) Add PBS to the liquid inlet and wash with shaking for 3 times, each time for 5 minutes; Step 5: After the chip is filled with PBS solution, imaging is performed; Step 6: Place positive and negative electrodes at the inlet and outlet, respectively, and energize the electrodes with a voltage of 3-16 volts for 1-5 minutes to ensure that sufficient active ingredients are generated near the positive electrode in the solution. Step 7: (1) Turn off the power to the electrode, add PBST solution to the liquid inlet to push the solution containing the active ingredient around the electrode and flow through the tissue slice area of ​​the chip, and then suck out the excess solution at the liquid outlet for 30-150 seconds; (2) Add PBST to the liquid inlet for washing three times, and finally fill it with PBST solution; Step 8: (1) After discarding the PBST solution in the chip, load an appropriate amount of elution solution preheated at 37°C into the liquid inlet to cover the tissue section area and leave it at room temperature for 3-5 minutes; (2) After discarding the eluent, reload an appropriate amount of 37°C preheated eluent from the liquid inlet, place the chip in a wet box at 37°C and incubate for 25-30 minutes. After incubation, wash with PBST three times, 3 minutes each time; Step 9: Repeat steps 4-5 for multiple rounds of labeling, staining, and imaging.

9. The device and method for cyclic staining of cells or tissues according to claim 8, characterized in that: The step 1 comprises the following steps: (1) Dewaxing and hydration ① Dewax the sections in xylene twice, 10 min each time; ② Then add anhydrous ethanol, anhydrous ethanol, 95% ethanol, and 85% ethanol in sequence and soak at room temperature for 5 minutes each time; ③ Rinse the slices twice with tap water to wash off the ethanol; (2) Antigen retrieval Select a repair solution suitable for the selected primary antibody to repair the tissue. First, boil it in a microwave at high temperature for 5 minutes, then immerse the slices in the repair solution and repair it at medium temperature in the microwave for 15 minutes, and cool it at room temperature for more than 2 hours.

10. The device and method for cyclic staining of cells or tissues according to claim 8, characterized in that: The specific operation of step 3 (2) is as follows: add an appropriate amount of PBST to the liquid inlet, let it stand for 1-3 minutes, and suck out the solution at the liquid outlet; repeat the above operation 3 times.

Images