Multi-immunofluorescent staining kit based on multi-antibody stable premixed solution as well as preparation method and application of multi-immunofluorescent staining kit

Abstract

The invention relates to the technical field of immunofluorescence detection, and particularly discloses a multiple immunofluorescence staining kit based on a multi-antibody stable premix solution as well as a preparation method and application of the multiple immunofluorescence staining kit. The kit comprises: a multi-antibody stable premix solution containing four fluorescence labeled antibodies targeting different antigens, each antibody being respectively coupled with a fluorescent dye of which the wavelength does not interfere with each other, and mixing the antibodies in a unified buffer system according to a preset proportion; the stable buffer system contains a phosphate buffer solution with the pH value of 7.2-7.6, trehalose, glycerol, bovine serum albumin and a nonionic surfactant; the antibody pretreatment unit comprises an antibody buffer replacement solution, a cross adsorbent and an Fc segment blocking antigen; and the sealing subpackaging pipe is used for low-temperature freeze-drying or liquid nitrogen cryopreservation. The kit can realize one-step co-staining of multi-target tissue slices, reduces operation steps, improves staining stability and signal specificity, and is suitable for pathological diagnosis, immune microenvironment analysis and biomedical research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunofluorescence detection technology, and specifically discloses a multiplex immunofluorescence staining kit based on a multi-antibody stable premix, a preparation method thereof and an application thereof. BACKGROUND

[0002] Multiplex immunofluorescence staining technology is an important biological analysis method, which is widely used in tumor diagnosis, histological research and spatial distribution analysis of immunological markers. Compared with the traditional single staining method, multiplex staining can simultaneously detect multiple targets in the same tissue section, greatly improving the efficiency and depth of information acquisition, and is especially suitable for the study of complex microenvironments such as tumor immune microenvironments. However, the existing technology often relies on manual antibody-by-antibody staining, which is tedious, has poor reproducibility, and has problems such as staining order and antibody interference, which limits its standardized application.

[0003] To solve the above problems, researchers have proposed a premix system in which multiple antibodies stably coexist, that is, multiple fluorescently labeled antibodies targeting different antigens are stably mixed in a predetermined ratio to prepare a ready-to-use staining solution. This strategy can theoretically simplify the staining process, reduce antibody usage and improve reproducibility. Existing premix systems still face technical bottlenecks such as antibody interference, signal attenuation and insufficient storage stability, especially in long-term storage or high-throughput use scenarios, making it difficult to guarantee the antigen binding activity and fluorescence signal consistency of the premix.

[0004] Therefore, it is of great significance to develop a new type of multiplex immunofluorescence staining kit. The kit should have the characteristics of antibody synergistic stability, non-crosstalk fluorescence signal, ready-to-use operation convenience, etc., and provide strong technical support for disease mechanism research and clinical auxiliary diagnosis. SUMMARY

[0005] To solve the above problems, the present application aims to provide a multiplex immunofluorescence staining kit based on a multi-antibody stable premix, which comprises: a. a multi-antibody stable premix containing four fluorescently labeled antibodies targeting different antigens, wherein the antibodies are coupled with fluorescent dyes of different wavelengths that do not interfere with each other, and are mixed in a unified buffer system at a predetermined concentration; b. a stable buffer system composed of a phosphate buffer solution (PBS) with a pH of 7.2-7.6, supplemented with 0.3-1.5% (w / v) trehalose, 5-15% (v / v) glycerol, 1-3% (w / v) bovine serum albumin (BSA), and 0.01-0.05% (v / v) non-ionic surfactant Tween-20; c. Antibody pretreatment unit, including antibody buffer replacement liquid and cross-adsorbent, the pretreatment liquid contains Tris-HCl, NaCl, BSA and Fc segment blocking antigen; d. Stable packaging structure, including low temperature freeze-drying or liquid nitrogen storage sealed sub-packaging tube, each sub-packaging tube contains the amount required for single staining.

[0006] As a preferred technical solution, the antibody comprises: Anti-human CD4 antibody: the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 1, and the corresponding nucleotide sequence is shown in SEQ ID NO: 9; the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2, and the corresponding nucleotide sequence is shown in SEQ ID NO: 10; Anti-human CD8 antibody: the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 3, and the corresponding nucleotide sequence is shown in SEQ ID NO: 11; the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 4, and the corresponding nucleotide sequence is shown in SEQ ID NO: 12; Anti-human PD-1 antibody: the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 5, and the corresponding nucleotide sequence is shown in SEQ ID NO: 13; the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 6, and the corresponding nucleotide sequence is shown in SEQ ID NO: 14; Anti-human Ki67 antibody: the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 7, and the corresponding nucleotide sequence is shown in SEQ ID NO: 15; the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 8, and the corresponding nucleotide sequence is shown in SEQ ID NO: 16.

[0007] As a preferred technical solution, the anti-human CD4 antibody is labeled with Alexa Fluor 488, with an emission wavelength of 519 nm; the anti-human CD8 antibody is labeled with Alexa Fluor 555, with an emission wavelength of 565 nm; the anti-human PD-1 antibody is labeled with Alexa Fluor 647, with an emission wavelength of 665 nm; and the anti-human Ki67 antibody is labeled with DyLight 405, with an emission wavelength of 421 nm.

[0008] As a preferred technical solution, the anti-human CD4 antibody is labeled with Cyanine 3, the emission wavelength is 570 nm; the anti-human CD8 antibody is labeled with Cyanine 5, the emission wavelength is 670 nm; the anti-human PD-1 antibody is labeled with Alexa Fluor 405, the emission wavelength is 421 nm; and the anti-human Ki67 antibody is labeled with Alexa Fluor 594, the emission wavelength is 617 nm.

[0009] As a preferred technical solution, for Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647 and DyLight 405, the coupling density of each fluorescent dye is controlled to be between 3 and 6 dye molecules per antibody molecule, the emission wavelengths of the dyes carried by each antibody are all greater than or equal to 25 nm different from those of the other three dyes, and the four fluorescent dyes have independent spectral responses in a PBS solution at pH 7.4.

[0010] As a preferred technical solution, the protein concentration of each fluorescently labeled antibody in the premix solution is 10-35 μg / mL, the total antibody protein concentration is 90-130 μg / mL, and the antibodies are mixed in a volume ratio of 1:1:1:1 to form a single liquid component, wherein the buffer is a PBS at pH 7.4, the liquid system is sterilized by a 0.22 μm filter membrane and is divided into 1 mL freeze-drying tubes.

[0011] As a preferred technical solution, the amino acid sequence of the Fc segment blocking the antigen is shown in SEQ ID NO: 17.

[0012] The application also provides a preparation method of the multiplex immunofluorescence staining kit, comprising the following steps: S1, antibody preparation: purifying the required monoclonal antibodies and enriching them to high purity by using protein A / G affinity chromatography; S2, fluorescent labeling: labeling the antibodies with NHS ester active fluorescent dyes, the dye / antibody molar ratio is controlled to be between 4:1 and 8:1, and free dyes are removed by gel permeation chromatography after the reaction; S3, buffer replacement and cross adsorption: uniformly replacing the buffer of the labeled antibodies with PBS at pH 7.4 containing 1% BSA, and adsorbing with human spleen extract proteins or miscellaneous proteins to remove potential cross-binding components; S4, compounding: mixing the antibodies of different fluorescent channels according to the preset ratio, adding trehalose, glycerol and surfactants to form the final stable system; S5. Quality Testing: Antibody labeling efficiency is detected using the dye / protein ratio; antigen binding activity is detected using ELISA; crosstalk of multi-channel fluorescence is detected using fluorescence confocal imaging; and stability is detected using accelerated aging. S6. Terminal dispensing: The qualified premixed liquid is poured into freeze-drying tubes or freeze dispensing tubes in equal portions, sealed, and then subjected to quick freezing or freeze-drying treatment at -80℃.

[0013] The present invention also provides a method for applying the aforementioned multiplex immunofluorescence staining kit in tissue section staining, comprising: A1. Tissue pretreatment: Tissue sections were dewaxed, hydrated and heat-induced antigen retrieval treatment, washed with PBS and then blocked with 5% normal goat serum. A2. Kit usage: Take the lyophilized or frozen premixed antibody solution, reconstitute it to the working concentration according to the ratio, add it dropwise to the tissue section, and incubate it in a humidified chamber at room temperature or 37°C for 30–60 minutes. A3. Washing and mounting: Rinse 3 times with PBS buffer, then add mounting medium containing DAPI to mount the slides. A4. Image Acquisition: Images were acquired using a multi-channel laser confocal microscope, and the intensity, location, and co-expression of each fluorescence signal were analyzed.

[0014] Beneficial effects: This invention avoids signal distortion problems caused by staining sequence, cross-interference, or antibody degradation in traditional multi-antibody stepwise staining by stably compounding four fluorescently labeled antibodies in a unified buffer system. In the premixed solution system, the antibodies are mixed in optimized proportions and combined with a stable system composed of trehalose, glycerol, BSA, and a nonionic surfactant, which enhances the structural integrity of the antibodies during low-temperature storage and lyophilization-reconstitution processes while ensuring antibody activity.

[0015] The fluorescently labeled antibodies used in this kit are controlled by dye density and optimized by spectral range. They have the characteristics of large emission wavelength intervals, no channel overlap, and stable dye signals. This effectively realizes high-resolution, multi-channel imaging under the condition of simultaneous staining of four targets, avoids misjudgment caused by fluorescence crosstalk, and enables one-step multi-target co-staining of tissue samples, shortening the staining cycle.

[0016] The Fc segment blocking antigen and cross-adsorbent introduced in the antibody pretreatment unit of this invention further reduce background noise and non-specific binding signals, improve the contrast of staining images and the accuracy of target identification, and provide a reliable, stable and efficient staining tool for immune microenvironment assessment, histopathological classification and drug action mechanism research. Attached Figure Description

[0017] Figure 1This is a schematic diagram illustrating the preparation method of the multiplex immunofluorescence staining kit of the present invention; Figure 2 This is a fluorescent staining photograph of Embodiment 1 of the present invention; Figure 3 This is a fluorescent staining photograph of Example 2 of the present invention. Detailed Implementation

[0018] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0019] Example 1 This embodiment provides a multiplex immunofluorescence staining kit based on a multi-antibody stable premix, the specific composition and parameters of which are as follows: a. Multi-antibody stable premix: Contains four human monoclonal antibodies targeting different antigens: anti-human CD4 antibody, anti-human CD8 antibody, anti-human PD-1 antibody, and anti-human Ki67 antibody. Each antibody is conjugated with a fluorescent dye to a concentration of 25 μg / mL, and the total protein concentration is 100 μg / mL. The four antibodies are mixed in a volume ratio of 1:1:1:1 to form a single liquid component.

[0020] Anti-human CD4 antibody was labeled with Alexa Fluor 488 (emission wavelength 519 nm), anti-human CD8 antibody was labeled with Alexa Fluor 555 (emission wavelength 565 nm), anti-human PD-1 antibody was labeled with Alexa Fluor 647 (emission wavelength 665 nm), and anti-human Ki67 antibody was labeled with DyLight 405 (emission wavelength 421 nm). The coupling density of each dye was controlled at 4 dye molecules per antibody molecule, and the emission wavelength interval between any two dyes was ≥25 nm.

[0021] b. Stabilizing buffer system: pH 7.4 phosphate buffer (PBS) with trehalose 0.8% (w / v), glycerol 10% (v / v), bovine serum albumin (BSA) 2% (w / v) and Tween-20 0.02% (v / v) added. The buffer is sterilized by filtration through a 0.22 μm membrane before use.

[0022] c. Antibody pretreatment unit: includes one vial of antibody buffer replacement solution (50 mM Tris-HCl, 0.15 M NaCl, BSA 2% w / v, pH 7.5) and one vial of cross-adsorbent, wherein the cross-adsorbent is 100 μg / vial of lyophilized human spleen extract total protein; and one vial of Fc segment blocking antigen (SEQ ID NO:9) solution, with a concentration of 1 mg / mL.

[0023] d. Stable packaging structure: All liquid components are dispensed into 1 mL lyophilized tubes, each containing 100 μL of the amount required for a single staining. After lyophilization, the tubes are sealed and stored at -20°C, with a shelf life of 6 months.

[0024] like Figure 1 As shown, the preparation process includes: S1. After purifying the four monoclonal antibodies by protein A / G affinity chromatography, they were replaced with pH 7.4 PBS (containing 1% BSA) and the concentration was adjusted to 1 mg / mL. S2. The antibody was reacted with NHS ester fluorescent dye solution (2 mg / mL, dissolved in DMSO) at a molar ratio of 4:1 for 30 min (room temperature, protected from light). The reaction solution was then subjected to Sephadex G-25 gel permeation chromatography to remove the free dye. S3. Mix the labeled antibody with human spleen extract protein at a ratio of 1:1 and adsorb for 20 min, then centrifuge to remove the precipitate. S4. Mix the four antibodies in a ratio of 1:1:1:1, add trehalose, glycerol, and Tween-20 to prepare a stable buffer system, and finally adjust it to the above concentration. S5, sterilized by 0.22 μm filter membrane, dispensed into freeze-drying tubes, freeze-dried for 48 h, and stored at -20℃.

[0025] How to use: Paraffin-embedded tissue sections (4 μm thick) of human tonsils were obtained, dried at 60℃ for 2 h, dewaxed (twice with xylene for 10 min each), and then subjected to gradient hydration (100%, 95%, 85%, and 75% ethanol for 5 min each, followed by rinsing with distilled water). The sections were then microwaved with citrate buffer (pH 6.0) for 15 min and rinsed three times with PBS.

[0026] Add 100 μL of the Fc blocking antigen solution from the antibody pretreatment unit of the multiplex immunofluorescence staining kit, incubate at room temperature for 15 min, aspirate excess liquid, then add 50 μL of the reconstituted multi-antibody stabilizing premix from the kit, and incubate at 37°C for 45 min in a humidified chamber. Wash three times with PBS (5 min each time), add mounting medium containing 1 μg / mL DAPI, and after mounting, use a multichannel laser confocal microscope to acquire images at excitation conditions of 405 nm, 488 nm, 555 nm, and 647 nm, respectively, and record the fluorescence signal intensity and target distribution. The fluorescence images are shown below. Figure 2 As shown.

[0027] Example 2 This embodiment provides a multiplex immunofluorescence staining kit based on a multi-antibody stable premix, the composition of which is as follows: a. Multi-antibody stable premix: Contains four human monoclonal antibodies targeting different antigens: anti-human CD4 antibody, anti-human CD8 antibody, anti-human PD-1 antibody, and anti-human Ki67 antibody. Each antibody is conjugated with a fluorescent dye to a concentration of 25 μg / mL, with a total protein concentration of 100 μg / mL, and is mixed in a volume ratio of 1:1:1:1 to form a single liquid component.

[0028] Anti-human CD4 antibody labeled Cyanine 3 (emission wavelength 570 nm); Anti-human CD8 antibody labeled Cyanine 5 (emission wavelength 670 nm); Anti-human PD-1 antibody labeled Alexa Fluor 405 (emission wavelength 421 nm); Anti-human Ki67 antibody labeled Alexa Fluor 594 (emission wavelength 617 nm).

[0029] The coupling density of each fluorescent dye is controlled at 4 dye molecules per antibody molecule, and the emission wavelength interval between any two dyes is ≥25 nm.

[0030] b. Stabilizing buffer system: pH 7.4 phosphate buffer (PBS) containing 0.8% (w / v) trehalose, 10% (v / v) glycerol, 2% (w / v) bovine serum albumin (BSA) and 0.02% (v / v) Tween-20, sterilized by a 0.22 μm filter membrane.

[0031] c. Antibody pretreatment unit: including antibody buffer replacement solution (50 mM Tris-HCl, 0.15 M NaCl, BSA 2%, pH 7.5), cross-adsorption agent (100 μg / bottle of lyophilized human spleen extract total protein) and Fc segment blocking antigen solution (SEQ ID NO: 9, 1 mg / mL).

[0032] d. Stable packaging structure: Each component of the kit is aliquoted into 1 mL lyophilized tubes, each containing the amount required for a single staining (100 μL), and stored at -20℃ for 6 months.

[0033] Preparation steps: S1. The four monoclonal antibodies were purified by protein A / G affinity chromatography, and the solution was changed to pH 7.4 PBS (containing 1% BSA) to adjust the concentration to 1 mg / mL. S2. Prepare NHS ester active fluorescent dyes (Cyanine 3, Cyanine 5, Alexa Fluor 405, Alexa Fluor 594, all 2 mg / mL, dissolved in DMSO), react at a dye / antibody molar ratio of 4:1 for 30 min (room temperature, protected from light), and remove free dyes by Sephadex G-25 gel permeation chromatography. S3. Mix the labeled antibody with human spleen extract protein at a 1:1 ratio and adsorb for 20 min, then centrifuge to remove the precipitate. S4. Mix the four antibodies in a 1:1:1:1 ratio, add trehalose, glycerol, and Tween-20 to form a stable buffer system, and adjust to the above concentration. S5. After sterilization by 0.22 μm filter membrane, dispensed into freeze-drying tubes, freeze-dried for 48 h, and stored at -20℃.

[0034] Application method: Paraffin-embedded sections (4 μm thick) from lung cancer patient tumor tissue were fixed in 10% neutral formalin for 24 h, then dehydrated and embedded. The sections were baked at 60℃ for 2 h, then dewaxed with xylene (twice, 10 min each time), gradient hydration (100%, 95%, 85%, 75% ethanol for 5 min each, followed by rinsing with distilled water), microwave retrieval with citrate buffer (pH 6.0) for 15 min, and rinsed three times with PBS.

[0035] Add 100 μL of the Fc blocking antigen solution from the antibody pretreatment unit in the kit, incubate at room temperature for 15 min, aspirate excess liquid, and then add 50 μL of the reconstituted multi-antibody stabilizing premix from the kit. Incubate in a humidified chamber at 37°C for 45 min. Wash three times with PBS (5 min each time), add mounting medium containing 1 μg / mL DAPI, and after mounting, acquire images using a multichannel laser confocal microscope at excitation conditions of 405 nm, 561 nm, 594 nm, and 647 nm to analyze the distribution of each fluorescence signal and co-expression, such as... Figure 3 As shown.

[0036] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multiplex immunofluorescence staining kit based on a multi-antibody stable premix, characterized in that, The kit includes: a. A multi-antibody stable premix containing four fluorescently labeled antibodies targeting different antigens, wherein each antibody is conjugated to a fluorescent dye with wavelengths that do not interfere with each other, and the antibodies are mixed in a uniform buffer system at a preset concentration; b. A stabilizing buffer system consisting of phosphate-buffered saline (PBS) at pH 7.2–7.6, supplemented with trehalose 0.3–1.5% (w / v), glycerol 5–15% (v / v), bovine serum albumin (BSA) 1–3% (w / v), and the nonionic surfactant Tween-20 0.01–0.05% (v / v). c. Antibody pretreatment unit, including antibody buffer replacement solution and cross-adsorption agent, the pretreatment solution contains Tris-HCl, NaCl, BSA and Fc segment blocking antigen; d. Stable packaging structure, including low-temperature freeze-drying or liquid nitrogen cryopreservation sealed dispensing tubes, each containing the amount required for a single dyeing session.

2. The multiplex immunofluorescence staining kit based on a multi-antibody stable premix according to claim 1, characterized in that, The antibodies include: Anti-human CD4 antibody: Its light chain variable region amino acid sequence is shown in SEQ ID NO:1, and the corresponding nucleotide sequence is shown in SEQ ID NO:9; its heavy chain variable region amino acid sequence is shown in SEQ ID NO:2, and the corresponding nucleotide sequence is shown in SEQ ID NO:

10. Anti-human CD8 antibody: Its light chain variable region amino acid sequence is shown in SEQ ID NO:3, and the corresponding nucleotide sequence is shown in SEQ ID NO:11; its heavy chain variable region amino acid sequence is shown in SEQ ID NO:4, and the corresponding nucleotide sequence is shown in SEQ ID NO:

12. Anti-human PD-1 antibody: Its light chain variable region amino acid sequence is shown in SEQ ID NO:5, and the corresponding nucleotide sequence is shown in SEQ ID NO:13; its heavy chain variable region amino acid sequence is shown in SEQ ID NO:6, and the corresponding nucleotide sequence is shown in SEQ ID NO:

14. Anti-human Ki67 antibody: Its light chain variable region amino acid sequence is shown in SEQ ID NO:7, and the corresponding nucleotide sequence is shown in SEQ ID NO:15; its heavy chain variable region amino acid sequence is shown in SEQ ID NO:8, and the corresponding nucleotide sequence is shown in SEQ ID NO:

16.

3. The multiplex immunofluorescence staining kit based on a multi-antibody stable premix according to claim 2, characterized in that, An anti-human CD4 antibody, labeled with Alexa Fluor 488, with an emission wavelength of 519 nm; An anti-human CD8 antibody, labeled with Alexa Fluor 555, with an emission wavelength of 565 nm; An anti-human PD-1 antibody, labeled with Alexa Fluor 647, with an emission wavelength of 665 nm; Anti-human Ki67 antibody, labeled with DyLight 405, with an emission wavelength of 421 nm.

4. The multiplex immunofluorescence staining kit based on a multi-antibody stable premix according to claim 2, characterized in that, Anti-human CD4 antibody, labeled with Cyanine 3, with an emission wavelength of 570 nm; Anti-human CD8 antibody, labeled with Cyanine 5, with an emission wavelength of 670 nm; An anti-human PD-1 antibody, labeled with Alexa Fluor 405, with an emission wavelength of 421 nm; An anti-human Ki67 antibody, labeled with Alexa Fluor 594, emits at a wavelength of 617 nm.

5. The multiplex immunofluorescence staining kit based on a multi-antibody stable premix according to claim 3 or 4, characterized in that, The coupling density of each fluorescent dye is controlled between 3 and 6 dye molecules per antibody molecule. The emission wavelength of each antibody-carried dye differs from that of the other three dyes by no less than 25 nm. Furthermore, the four fluorescent dyes have independent spectral responses in PBS solution at pH 7.

4.

6. The multiplex immunofluorescence staining kit based on a multi-antibody stable premix according to claim 1 or 2, characterized in that, The protein concentration of each fluorescently labeled antibody in the premix was 10–35 μg / mL, and the total antibody protein concentration was 90–130 μg / mL. The antibodies were mixed in a volume ratio of 1:1:1:1 to form a single liquid component. The buffer was phosphate-buffered saline (PBS) at pH 7.

4. The liquid system was sterilized by a 0.22 μm filter and dispensed into 1 mL lyophilized tubes.

7. The multiplex immunofluorescence staining kit based on a multi-antibody stable premix according to claim 5, characterized in that, The amino acid sequence of the Fc segment blocking antigen is shown in SEQ ID NO:

17.

8. A method for preparing a multiplex immunofluorescence staining kit as described in any one of claims 1-7, characterized in that, It includes the following steps: S1. Antibody preparation: Purify the required monoclonal antibody and enrich it with high purity using protein A / G affinity chromatography; S2. Fluorescent labeling: The antibody was labeled with NHS ester-active fluorescent dye, and the dye / antibody molar ratio was controlled between 4:1 and 8:

1. After the reaction, the free dye was removed by gel permeation chromatography. S3. Buffer Replacement and Cross-Adsorption: Replace the labeled antibody buffer with PBS at pH 7.4 containing 1% BSA, and adsorb human spleen extract protein or other proteins to remove potential cross-binding components. S4. Compounding: Mix antibodies with different fluorescent channels according to a preset ratio, and add trehalose, glycerol and surfactants to form a final stable system; S5. Quality Inspection: Antibody labeling efficiency is detected using the dye / protein ratio; Antigen binding activity was detected using ELISA; crosstalk of multi-channel fluorescence was detected using fluorescence confocal imaging; and stability was detected using accelerated aging assay. S6. Terminal dispensing: The qualified premixed liquid is poured into freeze-drying tubes or freeze dispensing tubes in equal portions, sealed, and then subjected to quick freezing or freeze-drying treatment at -80℃.

9. A method for using the multiplex immunofluorescence staining kit as described in any one of claims 1-7 in tissue section staining, characterized in that, include: A1. Tissue pretreatment: Tissue sections were dewaxed, hydrated and heat-induced antigen retrieval treatment, washed with PBS and then blocked with 5% normal goat serum. A2. Kit usage: Take the lyophilized or frozen premixed antibody solution, reconstitute it to the working concentration according to the ratio, add it dropwise to the tissue section, and incubate it in a humidified chamber at room temperature or 37°C for 30–60 minutes. A3. Washing and mounting: Rinse 3 times with PBS buffer, then add mounting medium containing DAPI to mount the slides. A4. Image Acquisition: Images were acquired using a multi-channel laser confocal microscope, and the intensity, location, and co-expression of each fluorescence signal were analyzed.

Images