Multi-histochemical staining method based on controllable staining-decolorizing circulation

Abstract

The invention relates to the technical field of biomedical detection, and particularly discloses a multiple histochemical staining method based on controllable staining-decolorizing circulation. The method is realized through the steps of water bath-assisted antigen repair, composite pretreatment, alternate labeling dyeing of nanogold and quantum dots, electrochemical-assisted controllable decoloration, dynamic monitoring circulation, optical transparency treatment, multi-modal imaging reconstruction and the like. The key composition comprises a composite decolorizing agent containing a specific electron shuttling medium and a series of coloring agents containing nanogold and quantum dots. The preparation method mainly comprises the following steps: performing incubation reaction on each component and a tissue sample according to a specific sequence, completing controllable decolorization circulation under the assistance of an electric field, and regulating and controlling the process in real time through an optical monitoring system. According to the method disclosed by the invention, the sequential marking and accurate recognition of multiple target molecules on the same tissue sample can be realized; in addition, the preparation method has the advantages of being high in process controllability, good in dyeing cycle stability and high in operation repeatability.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, and more specifically, it relates to a multiple histochemical staining method based on a controlled staining-decolorization cycle. Background Technology

[0002] In biomedical fields such as pathological diagnosis and tumor molecular mechanism research, the simultaneous localization and correlation analysis of multiple target molecules in tissue samples is crucial for revealing the patterns of disease development and guiding the formulation of clinical treatment plans. Because clinical biopsy samples are often few and precious, traditional single-round histochemical staining techniques require multiple identical samples, leading not only to sample waste but also to potential deviations in test results due to differences in sample processing.

[0003] However, existing multiple cycle staining techniques face core bottlenecks in practical applications: On the one hand, the tissue fixation step generally uses a single paraformaldehyde fixative, lacking components that can synergistically cross-link with it. This leads to the tissue becoming structurally loose, with deformed cell morphology, or even detached during repeated staining-decolorization chemical reagent soaking and treatment, failing to maintain the stable structural basis required for subsequent staining. On the other hand, the decolorization step relies heavily on a single chemical decolorizing agent, lacking both a composite system containing specific electron shuttle media to improve decolorization efficiency and electric field-assisted control methods. At the same time, the labeling methods do not form an efficient alternation, making it difficult to completely remove the staining signal from the previous round. During subsequent staining, the residual signal and the newly labeled signal superimpose and interfere with each other, ultimately failing to achieve the detection goal of multiple cycle stainings on the same tissue sample with clear and interference-free signals in each round, severely limiting the application of this technology in multi-molecule synergistic analysis scenarios. Summary of the Invention

[0004] To address the problems of severe signal interference and limited cycle number in existing multiple staining techniques, this application provides a multiple histochemical staining method based on controllable staining-decolorization cycles.

[0005] A multiple histochemical staining method based on a controlled staining-destaining cycle includes the following steps:

[0006] S1. Water bath-assisted antigen retrieval: Water bath-assisted antigen retrieval: Using EDTA alkaline retrieval solution, tissue samples are treated with water bath-assisted antigen retrieval. The retrieval temperature range is 95-100℃, and the retrieval time range is 20-40min.

[0007] S2. Composite pretreatment: Composite pretreatment is performed on the repaired tissue samples. First, the samples are fixed with a mixed fixative for 2-8 hours, followed by gradient dehydration, paraffin embedding, and sectioning. The section thickness ranges from 3 to 8 μm.

[0008] S3, First round of nano-gold labeling staining: Perform the first round of staining by incubating the tissue sample with the first staining agent at a temperature of 22-35℃ for 15-50 minutes;

[0009] S4. Electrochemical-assisted controlled decolorization: Perform the first round of controlled decolorization by treating tissue samples with a composite decolorizing agent. During the treatment, control the pH value to 6.2-7.8, the treatment time to 8-25 min, and the solution oxidation-reduction potential to -200 mV to +100 mV.

[0010] S5. Second round of quantum dot labeling staining: Perform the second round of staining by incubating the destained tissue sample with the second staining agent at a temperature of 22-35℃ for 15-50 minutes.

[0011] S6. Dynamic monitoring of cyclic staining: Repeat steps S4 and S5, each time using a different staining agent to label different target molecules. The number of repetitions ranges from 1 to 4 times, and the destaining efficiency is evaluated using a dynamic optical monitoring system after each cycle.

[0012] S7. Optical Transparency Treatment: A mixed solution containing urea and glycerol is used as an optical transparentizing agent to perform optical transparency treatment on multiple stained tissue samples;

[0013] S8. Multimodal Imaging Reconstruction: First, multimodal imaging analysis is performed on the processed tissue sample to obtain fluorescence images, dark-field images, and confocal images; then, based on the obtained images, three-dimensional reconstruction is performed using an image fusion algorithm.

[0014] By employing the above technical solution, using EDTA alkaline retrieval solution as the antigen retrieval solution, and controlling the water bath temperature at 95-100℃ and the time at 20-40 min, the antigen epitopes in the tissue sample are gently and uniformly exposed, maintaining the integrity of the antigen structure. This ensures stable binding of antibodies and antigens during subsequent multiple staining. The retrieval tissue samples are fixed with a mixed fixative solution for 2-8 hours, followed by gradient dehydration, paraffin embedding, and sectioning to a thickness of 3-8 μm. This maintains the original morphology and structure of the tissue sample, prevents shrinkage or fragmentation during subsequent processing, and ensures uniform section thickness, thus facilitating subsequent staining. The process achieves uniform penetration of the staining agent into the tissue and clear observation during imaging. By incubating the tissue sample with a first staining agent containing a specific antibody against the first target molecule, a secondary antibody labeled with gold nanoparticles, and a horseradish peroxidase substrate system at 22-35℃ for 15-50 min, the antibody in the first staining agent specifically binds to the first target molecule. Simultaneously, the labeling characteristics of gold nanoparticles and the signal amplification effect of the horseradish peroxidase substrate system are utilized to achieve precise labeling and signal enhancement of the first target molecule. The tissue sample is treated with a composite destaining agent containing dithiothreitol, electron shuttle medium, and buffer solution, while controlling the pH value at 6.2-7 during the treatment process.8. The staining time is 8-25 min, and the solution redox potential is between -200 mV and +100 mV. This process specifically removes the staining agent components bound in the first round of staining, avoids interference from residual staining agent in subsequent staining, and ensures controllable decolorization through parameter adjustment, thus providing a clean tissue sample background for the second round and subsequent cycles of staining. The decolorized tissue sample is then incubated with a second staining agent containing a specific antibody against the second target molecule, a quantum dot-labeled secondary antibody, and a fluorescent dye at 22-35℃ for 15-50 min. This allows the antibody in the second staining agent to specifically bind to the second target molecule, and leverages the fluorescence properties of quantum dots and the auxiliary labeling effect of the fluorescent dye to achieve precise labeling and fluorescence signal presentation of the second target molecule. The electrochemically assisted controllable decolorization and targeted staining steps are repeated 1-4 times, and the decolorization effect is evaluated after each cycle using a dynamic optical monitoring system including a spectrometer and a CCD detector. The system plays a crucial role in adjusting subsequent decolorization parameters based on monitoring results, ensuring thorough removal of the previous staining agent and effective binding of the new staining agent, thereby enabling sequential labeling of multiple target molecules and expanding the coverage of multiple staining. By using a mixed solution containing urea and glycerol as an optical clearing agent to process the multiple-stained tissue samples, the system reduces light scattering, enhances light penetration, and prevents tissue deformation or loss of staining signals, thus providing favorable optical conditions for subsequent multimodal imaging. Multimodal imaging analysis of the cleared tissue samples using fluorescence, dark-field, and confocal images, followed by 3D reconstruction using image fusion algorithms, integrates target molecule signals and tissue structure information from different imaging methods, compensating for the limitations of single imaging methods, and ultimately achieving a three-dimensional representation of the spatial distribution of multiple target molecules and the tissue structure within the tissue sample.

[0015] Preferably, in step S1, the tissue sample is formalin-fixed paraffin-embedded tissue or frozen tissue.

[0016] By adopting the above technical solutions, the samples can be adapted to common clinical pathological sample processing scenarios. Formalin-fixed paraffin-embedded tissue is a routine sample type for pathological diagnosis, which can still perform effective antigen retrieval after long-term storage. Frozen tissue can better preserve the active ingredients and easily degradable antigens in the sample, meeting the needs of fresh samples or specific sensitive antigen detection. This provides a suitable sample basis for subsequent water bath-assisted antigen retrieval steps, ensuring that the retrieval process can be carried out stably according to the characteristics of the sample type, and avoiding the problem of poor retrieval effect due to unclear sample type.

[0017] Preferably, in step S2, the mixed fixative is a mixture of paraformaldehyde and glutaraldehyde, wherein the concentration of paraformaldehyde ranges from 3 to 6 wt% and the concentration of glutaraldehyde ranges from 0.5 to 2 wt%.

[0018] By employing the above technical solution, the synergistic effect of using paraformaldehyde to cross-link tissue proteins to maintain overall structural stability, and leveraging the dialdehyde properties of glutaraldehyde to precisely cross-link microstructures within the tissue, is achieved. The concentration range of paraformaldehyde avoids insufficient fixation due to excessively low concentration leading to tissue dissolution, or excessive hardening of the tissue due to excessively high concentration. The concentration range of glutaraldehyde enhances the fixation effect of fine structures while reducing the destruction of subsequent antigen site activity. This provides a stable tissue basis for the complete preservation of tissue morphology during subsequent gradient dehydration and paraffin embedding, as well as the effective binding of staining agents to target molecules after sectioning. This ensures that the pretreated tissue sample has both good structural integrity and maintains the recognizableness of the target antigen.

[0019] Preferably, in step S3, low-frequency ultrasound is applied during the incubation process, with an ultrasound frequency range of 20-40kHz and a power density range of 0.5-2W / cm².

[0020] By adopting the above technical solution, the mechanical vibration effect of low-frequency ultrasound is used to accelerate the penetration and diffusion of the first staining agent in the tissue sample. The frequency range of 20-40kHz can effectively act on the interstitial tissue, pushing the staining agent molecules to break through the tissue barrier and avoiding the local overheating problem that is prone to occur with high-frequency ultrasound. The power density of 0.5-2W / cm² can ensure the diffusion efficiency of the staining agent while preventing the damage to the antibody activity in the staining agent or the structural damage to the tissue sample caused by excessive power. This creates conditions for the specific antibody in the first staining agent to bind rapidly and uniformly with the first target molecule in the tissue, ensuring that the incubation process is fully completed within the set time, reducing the difference in signal intensity caused by uneven staining agent penetration, and providing a guarantee for the stable presentation of subsequent staining signals.

[0021] Preferably, in step S3, the first staining agent comprises a specific antibody against the first target molecule, a secondary antibody labeled with gold nanoparticles, and a horseradish peroxidase substrate system, wherein the gold nanoparticles have a particle size range of 5-20 nm and a concentration of 0.01-0.5 mg / mL.

[0022] By employing the above technical solution, a multi-component synergistic approach is achieved to precisely label and enhance the signal of the primary target molecule. Specifically, the antibody targeting the primary target molecule accurately identifies and binds to it within the tissue, providing a targeting basis for subsequent labeling. The secondary antibody labeled with gold nanoparticles, through specific binding with the specific antibody, anchors the gold nanoparticles at the target molecule. The 5-20 nm particle size range ensures good tissue penetration, allowing the gold nanoparticles to successfully reach the target site within the tissue, while also generating a stable and detectable optical signal, such as the scattering signal in dark-field imaging. This avoids insufficient signal intensity due to excessively small particle size or excessively large particle size. It significantly hinders tissue penetration; the concentration range of 0.01-0.5 mg / mL ensures a sufficient number of gold nanoparticles to label the target molecule and guarantee signal measurability, while reducing background interference caused by the non-specific adsorption of excess gold nanoparticles on the tissue surface or in the gaps; the horseradish peroxidase substrate system further amplifies the labeling signal of the target molecule by reacting with horseradish peroxidase conjugated to the secondary antibody, thereby providing a labeling basis with strong targeting, stable signal and low background interference for the subsequent clear detection of the primary target molecule, avoiding the problem of blurred target molecule recognition caused by missing labeling components, improper gold nanoparticle parameters or insufficient signal amplification.

[0023] Preferably, the horseradish peroxidase substrate system is an enhanced chemiluminescent substrate containing luminol and hydrogen peroxide, wherein the concentration of luminol ranges from 0.5 to 10 mmol / L and the concentration of hydrogen peroxide ranges from 0.01 to 0.1 wt%.

[0024] By employing the above technical solution, the synergistic effect of luminol as the core substance for chemiluminescence and hydrogen peroxide as the oxidant is achieved. The concentration range of luminol (0.5-10 mmol / L) ensures a sufficiently strong luminescence signal under horseradish peroxidase catalysis. A concentration as low as 0.5 mmol / L meets the signal detection requirements for low-expression target molecules, while a concentration up to 10 mmol / L is suitable for high-expression target molecules and avoids non-specific background luminescence caused by excessively high concentrations. It also avoids weak luminescence signals that are difficult to capture due to insufficient concentration. The concentration range of hydrogen peroxide (0.01-0.1 wt%) effectively facilitates the enzymatic reaction. Oxidation conditions should be provided, with 0.01 wt% gently initiating the reaction and 0.1 wt% improving reaction efficiency. This prevents excessive concentrations from damaging horseradish peroxidase activity or causing excessive oxidative damage to tissue samples. Furthermore, by leveraging the characteristics of enhanced chemiluminescent substrates and precisely controlling the concentrations of luminol and hydrogen peroxide, a stable and efficient signal amplification basis is provided for the horseradish peroxidase catalytic reaction. This ensures that the first target molecule labeled in the first staining agent can be identified through a clear and measurable chemiluminescent signal, avoiding problems such as insufficient signal amplification, severe background interference, or impaired enzyme activity caused by improper substrate composition or concentration imbalance.

[0025] Preferably, in step S4, the composite decolorizing agent comprises dithiothreitol, an electron shuttle medium, and a buffer solution. The electron shuttle medium is a ruthenium bipyridine complex or a ruthenium terpyridine complex with a concentration range of 0.5-3 mmol / L. A weak electric field is applied during the decolorization process, with an electric field strength range of 0.5-2 V / cm.

[0026] By employing the above technical solution, a multi-component and physical field synergistic effect is achieved to realize controllable decolorization. Dithiothreitol breaks the disulfide bonds formed during the binding of the staining agent and the target molecule, providing a chemical basis for the staining agent's detachment from the tissue sample. Ruthenium bipyridine complexes or ruthenium terpyridine complexes, acting as electron shuttle media, efficiently transfer electrons to promote the redox reaction of the staining agent components, accelerating the degradation or detachment of the staining agent. A concentration range of 0.5-3 mmol / L ensures that the electron shuttle media possesses basic electron transfer efficiency to drive the decolorization reaction, while avoiding excessively high concentrations that could cause non-specific redox reactions that damage the tissue sample or interfere with subsequent staining agent activity. The buffer solution maintains the decolorization system. The pH value is stabilized within the range of 6.2-7.8 set in step S4 to prevent pH fluctuations from affecting the activity of the destaining agent and the structural stability of the tissue sample. The weak electric field of 0.5-2V / cm can directionally drive the components of the composite destaining agent to move towards the staining residue site in the tissue, improving the contact efficiency between the destaining agent and the residual staining agent. At the same time, it avoids the electric field strength being too high and damaging the tissue sample structure, or too low and failing to achieve the directional driving effect. Thus, through the synergistic effect of chemical components and physical field, it ensures that the staining agent residue from the first round of staining is fully and specifically removed, providing a clean tissue background for subsequent cyclic staining steps, and avoiding problems such as interference with subsequent staining signals and sample rejection caused by incomplete destaining, low destaining efficiency, or tissue damage.

[0027] Preferably, in step S5, the second staining agent comprises a specific antibody against the second target molecule, a quantum dot-labeled secondary antibody, and a fluorescent dye, wherein the quantum dot particle size ranges from 3 to 15 nm, the emission wavelength is tunable in the range of 450 to 650 nm, and the quantum dot-labeled secondary antibody is coupled to the antibody via a click chemistry reaction. The click chemistry reaction uses a combination of cyclooctyne and azide, and the reaction time ranges from 30 to 90 min.

[0028] By employing the above technical solution, a multi-component synergistic approach is achieved to precisely label and adapt the second target molecule to the signal. Specifically, the antibody targeting the second target molecule can bind directionally to the target site within the tissue, providing a targeted anchor for labeling; the quantum dots' 3-15nm particle size range ensures smooth penetration into the interstitial space to reach the target site while maintaining stable fluorescence emission characteristics; the tunable emission wavelength of 450-650nm adapts to different detection channel requirements, avoiding interference caused by overlapping signal wavelengths with the first staining agent or subsequent cyclic staining agents; the fluorescent dye assists the quantum dots in enhancing the labeling signal, improving the detectability of low-expression target molecules; and the click chemistry reaction of cyclooctyne and azide allows for efficient coupling of quantum dots without the need for a metal catalyst. The secondary antibody avoids the destruction of antibody activity or contamination of tissues by metal ions. The reaction time range of 30-90 minutes allows for rapid conjugation, while a longer time of 90 minutes ensures sufficient conjugation. This avoids low conjugation efficiency and weak labeling signal due to excessively short time, or non-specific conjugation due to excessively long time. The buffer provides a suitable environment for the conjugation reaction and staining process, avoiding the impact of pH fluctuations on antibody binding capacity or quantum dot fluorescence characteristics. This provides a basis for highly targeted, distinguishable, and stable labeling efficiency for the specific labeling of the second target molecule, avoiding problems such as blurred target molecule recognition, signal interference, or labeling failure caused by missing labeling components, inappropriate quantum dot parameters, inefficient conjugation methods, or unbalanced reaction time.

[0029] Preferably, in step S6, the dynamic optical monitoring system includes a spectrometer and a CCD detector, with a monitoring wavelength range of 400-700nm, and automatically adjusts the subsequent decolorization time according to the monitoring results, with an adjustment time range of 1-10min.

[0030] By adopting the above technical solution, the decolorization effect can be accurately evaluated from multiple dimensions and the decolorization parameters can be dynamically optimized. The spectrometer can analyze the light absorption or emission spectrum in the range of 400-700nm to quantitatively detect the characteristic signal intensity of residual staining agents in tissue samples, thereby achieving a numerical evaluation of decolorization efficiency. The CCD detector can simultaneously capture optical images of tissue samples, intuitively presenting the spatial distribution of residual staining agents, avoiding the problem of ignoring local residues in simple spectral analysis. The wavelength range of 400-700nm covers the characteristic signal range of common staining agents, ensuring that all kinds of residual signals can be effectively monitored. The automatic adjustment range of 1-10min can flexibly increase or decrease the subsequent decolorization time according to the monitored residual amount, avoiding incomplete decolorization due to too small an adjustment range or tissue damage and wasted time due to too large an adjustment range. This provides real-time and accurate feedback for each staining-decolorization cycle, ensuring that the staining residue of the previous round is fully removed, creating a clean tissue background for the next round of staining, and avoiding problems such as interference and low efficiency of cyclic staining signals caused by inaccurate decolorization evaluation or fixed parameters.

[0031] Preferably, in step S7, the urea concentration ranges from 20-40 wt%, the glycerol concentration ranges from 30-50 wt%, and the treatment time is 1-6 h.

[0032] By employing the above technical solution, the synergistic effect of urea and glycerol is utilized to optimize tissue transparency. Urea can reduce the refractive index difference within the tissue by disrupting hydrogen bonds between tissue proteins. A concentration range of 20-40 wt% can effectively break down the dense structure of the tissue to improve light transmittance while avoiding excessive tissue swelling or protein denaturation due to excessive concentration. Glycerol, as a high refractive index substance, can form an affinity with tissue components to regulate the overall refractive index. A concentration range of 30-50 wt% can enhance the penetration of light into the tissue while reducing tissue dehydration and shrinkage caused by excessive concentration. A processing time of 1-6 hours ensures that the clearing agent fully penetrates all layers of the tissue, avoiding incomplete transparency and severe light scattering due to too short a time, or loss of staining signal due to prolonged immersion due to too long a time. This provides optically stable tissue samples for subsequent multimodal imaging, ensuring that fluorescence, dark-field, and confocal images can clearly present target molecular signals and tissue structural details, avoiding problems such as image blurring and signal attenuation caused by improper proportions of clearing agent components or imbalanced processing time.

[0033] In summary, this application has the following beneficial effects:

[0034] 1. Since this application uses a mixed fixative system containing paraformaldehyde and glutaraldehyde, and glutaraldehyde can produce a synergistic cross-linking effect with paraformaldehyde, the stability of the tissue structure is enhanced. At the same time, by alternating the use of nano-gold labeling and quantum dot labeling, combined with a composite decolorizing agent containing a specific electron shuttle medium and electric field-assisted decolorization technology, the technical effect of achieving multiple cycles of staining on the same tissue sample without interference between signals in each cycle is obtained.

[0035] 2. In this application, a low-frequency ultrasound-assisted incubation process is preferred. Ultrasound in a specific frequency range can promote the penetration and diffusion of staining agent molecules inside the tissue. At the same time, a dynamic optical monitoring system is used to detect the decolorization process in real time and automatically adjust the processing time according to the spectral changes. This achieves the technical effect of improving staining efficiency and ensuring that the cyclic staining process is stable and controllable.

[0036] 3. The method of this application uses a urea and glycerol mixed solution of a specific concentration ratio for optical transparency treatment. Since this ratio can effectively match the refractive index of the tissue and the transparent agent, combined with the simultaneous acquisition of fluorescence, dark field and confocal image data by a multimodal imaging system, and the use of image fusion algorithm for three-dimensional reconstruction, the technical effect of improving the imaging quality of deep tissue and realizing multi-dimensional structural analysis is achieved. Attached Figure Description

[0037] Figure 1 This is a flowchart of a multiple histochemical staining method based on a controlled staining-decolorization cycle provided in this application. Detailed Implementation

[0038] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0039] Technical concept:

[0040] In existing multiple cycle staining techniques, it is difficult to achieve multiple cycles of staining for the same tissue sample without interference between signals. The core reasons are twofold: First, tissue fixation relies on a single paraformaldehyde, which lacks components that can produce synergistic cross-linking effects. After multiple staining-decolorization processes, the tissue is prone to loosening and deformation due to insufficient structural stability, thus damaging the basis for subsequent staining. Second, the decolorization process uses a single chemical reagent, which lacks an efficient electron transport system and physical field regulation. Furthermore, the labeling method does not form an orderly alternation, resulting in residual staining signals from the previous round that interfere with the signals of subsequent new labels.

[0041] This technical solution specifically employs a series of techniques to address the aforementioned issues: synergistic cross-linking is achieved through a mixed fixative of paraformaldehyde and glutaraldehyde, enhancing the stability of the tissue structure during multiple cycles; alternating use of nano-gold labeling and quantum dot labeling, combined with a composite decolorizing agent containing ruthenium bipyridine or ruthenium terpyridine complexes and electric field-assisted decolorization, improves decolorization efficiency and thoroughness; simultaneously, a dynamic optical monitoring system is introduced to adjust decolorization parameters in real time, ensuring that the staining signals of each round do not interfere with each other, ultimately achieving multiple controllable cyclic staining cycles on the same tissue sample.

[0042] Preparation Example 1

[0043] The preparation method of the electron shuttle medium, ruthenium bipyridine complex, is as follows:

[0044] Take 5 mmol of ruthenium trichloride hydrate (RuCl3・3H2O) and add it to 50 mL of a 3:1 (v / v) mixture of ethanol and water, stirring until completely dissolved. Separately, take 15 mmol of 2,2'-bipyridine, dissolve it in 20 mL of ethanol, and slowly add it dropwise to the above ruthenium trichloride solution under nitrogen protection. After the addition is complete, raise the temperature to 80 °C and stir under reflux for 6 hours. After the reaction is complete, cool to room temperature, remove about 60% of the solvent by rotary evaporation, add 30 mL of saturated potassium hexafluorophosphate aqueous solution, and stir for 30 minutes until a large amount of orange-red solid precipitates. Collect the solid by filtration, wash twice with 50 mL of ice water to remove residual salts, and then wash once with 20 mL of diethyl ether to remove organic impurities. Dissolve the obtained solid in a small amount of acetonitrile, load it onto a silica gel column, and perform chromatography with acetonitrile:water = 9:1 as the eluent. Collect the second orange-red elution peak, evaporate under reduced pressure to remove the solvent, and dry under vacuum for 4 hours to obtain the ruthenium bipyridine complex (Ru(bpy)). 32 Solid, purity ≥98% as determined by HPLC.

[0045] Preparation Example 2

[0046] The preparation method of quantum dot-labeled secondary antibodies is as follows:

[0047] Take 10 mL of 2 μmol / L oil-phase CdSe / ZnS quantum dots (3-15 nm particle size), add 5 mL of ethanol solution containing 5% oleic acid, stir for 10 minutes, then add 20 mL of 0.1 M PBS buffer (pH 6.5), sonicate for 30 minutes, centrifuge at 8000g for 10 minutes to collect the aqueous phase, and obtain water-soluble quantum dots with carboxyl groups on the surface. Add 10 mmol EDC and 8 mmol NHS to the above quantum dot solution, activate at room temperature for 30 minutes, then add 5 μmol of diphenylcyclooctyne-polyethylene glycol-amino (95% purity), stir at 25°C in the dark for 2 hours, and filter through a 0.22 μm filter membrane to obtain cyclooctyne-functionalized quantum dots.

[0048] Take another 2 mg of secondary antibody, dissolve it in 1 mL of 0.1 M PBS (pH 7.4), add 10 μmol of azido-succinimide ester, and incubate overnight at 4 °C in the dark. Transfer the reaction solution to a 3 kDa dialysis bag and dialyze it in 0.01 M PBS (pH 7.2) for 24 hours, changing the solution 3 times to obtain the azido-modified secondary antibody.

[0049] Cyclooctyne-functionalized quantum dots were mixed with azide-modified secondary antibody at a molar ratio of 5:1, and 0.1M PBS was added to a total volume of 2 mL. The mixture was incubated at 25°C in the dark for 30-90 minutes. The reaction was carried out using a Superdex 200 size exclusion column with 0.01M PBS as the eluent at pH 7.4. The first fluorescence peak fraction was collected, which was the quantum dot-labeled secondary antibody. The concentration was adjusted to 0.8 mg / mL and stored at 4°C in the dark.

[0050] The following are the main raw materials and reagents used in the preparation examples, embodiments, and comparative examples, and their sources and specifications are as follows; unless otherwise specified, all reagents are commercially available analytical grade or higher products:

[0051] 1. Paraformaldehyde was purchased from Shandong Yifei Science and Trade Co., Ltd., CAS: 30525-89-4;

[0052] 2. Glutaraldehyde was purchased from Wuhan Jiyesheng Chemical Co., Ltd., CAS: 111-30-8;

[0053] 3. Luminol was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S19197;

[0054] 4. Dithiothreitol was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S64054;

[0055] 5. 2,2'-Bipyridine was purchased from Hubei Jusheng Technology Co., Ltd., product number: JS4028;

[0056] 6. Diphenylcyclooctyne-polyethylene glycol-amino was purchased from Guangzhou Carbon Technology Co., Ltd., purity: 95%;

[0057] 7. Azide-succinimide ester was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: Y63852.

[0058] Example 1

[0059] This application provides a multiple histochemical staining method based on a controlled staining-destaining cycle, comprising the following steps:

[0060] S1. Water bath-assisted antigen retrieval: Water bath-assisted antigen retrieval: Using EDTA alkaline retrieval solution, tissue samples are treated with water bath-assisted antigen retrieval. The retrieval temperature range is 98℃, and the retrieval time range is 30min.

[0061] Among them, the tissue samples were formalin-fixed paraffin-embedded tissues;

[0062] S2. Composite pretreatment: Composite pretreatment was performed on the repaired tissue samples. First, the samples were fixed with a mixed fixative for 5 hours, followed by gradient dehydration, paraffin embedding, and sectioning. The section thickness ranged from 5.5 μm.

[0063] The mixed stationary solution was a mixture of paraformaldehyde and glutaraldehyde, with paraformaldehyde concentration ranging from 4.5 wt% and glutaraldehyde concentration ranging from 1.25 wt%.

[0064] S3, First round of nano-gold labeling staining: Perform the first round of staining by incubating the tissue sample with the first staining agent at a temperature of 28.5℃ for 32.5 min;

[0065] Among them, low-frequency ultrasound assistance is applied during the incubation process, with an ultrasound frequency range of 30kHz and a power density range of 1.25W / cm².

[0066] The first staining agent contains a specific antibody against the first target molecule, a secondary antibody labeled with gold nanoparticles, and a horseradish peroxidase substrate system. The gold nanoparticles have a particle size range of 12.5 nm and a concentration of 0.255 mg / mL.

[0067] The horseradish peroxidase substrate system is an enhanced chemiluminescent substrate comprising luminol and hydrogen peroxide, with luminol concentrations ranging from 5.25 mmol / L to 0.055 wt%.

[0068] S4. Electrochemical-assisted controlled decolorization: The first round of controlled decolorization is carried out by treating tissue samples with a composite decolorizing agent. During the treatment, the pH value is controlled at 7.0, the treatment time is 16.5 min, and the redox potential of the solution is -50 mV.

[0069] The composite decolorizing agent contains dithiothreitol, an electron shuttle medium, and a buffer solution. The electron shuttle medium is a ruthenium bipyridine complex with a concentration range of 1.75 mmol / L. A weak electric field is applied during the decolorization process, with an electric field strength range of 1.25 V / cm.

[0070] S5. Second round of quantum dot labeling staining: Perform the second round of staining by incubating the destained tissue sample with the second staining agent at a temperature of 28.5℃ for 32.5 min.

[0071] The second staining agent contains a specific antibody against the second target molecule, a quantum dot-labeled secondary antibody, and a fluorescent dye. The quantum dot particle size ranges from 9 nm, and the emission wavelength is adjustable from 550 nm. The quantum dot-labeled secondary antibody is coupled to the antibody via a click chemistry reaction. The click chemistry reaction uses a combination of cyclooctyne and azide, and the reaction time ranges from 60 min.

[0072] S6. Dynamic monitoring of cyclic staining: Repeat steps S4 and S5, each time using a different staining agent to label different target molecules. The number of repetitions is up to 2, and the destaining efficiency is evaluated using a dynamic optical monitoring system after each cycle.

[0073] The dynamic optical monitoring system used includes a spectrometer and a CCD detector, with a monitoring wavelength range of 550 nm, and automatically adjusts the subsequent decolorization time based on the monitoring results, with an adjustment time range of 5.5 min.

[0074] S7. Optical Transparency Treatment: A mixed solution containing urea and glycerol is used as an optical transparentizing agent to perform optical transparency treatment on multiple stained tissue samples;

[0075] The urea concentration range was 30 wt%, the glycerol concentration range was 40 wt%, and the treatment time was 3.5 h.

[0076] S8. Multimodal Imaging Reconstruction: First, multimodal imaging analysis is performed on the processed tissue sample to obtain fluorescence images, dark-field images, and confocal images; then, based on the obtained images, three-dimensional reconstruction is performed using an image fusion algorithm.

[0077] Example 2

[0078] This application provides a multiple histochemical staining method based on a controlled staining-destaining cycle, comprising the following steps:

[0079] S1. Water bath-assisted antigen retrieval: Water bath-assisted antigen retrieval: Using EDTA alkaline retrieval solution, tissue samples are treated with water bath-assisted antigen retrieval. The retrieval temperature range is 95℃, and the retrieval time range is 20min.

[0080] Among them, the tissue samples were frozen tissues;

[0081] S2. Composite pretreatment: Composite pretreatment was performed on the repaired tissue samples. First, the samples were fixed with a mixed fixative for 2 hours, followed by gradient dehydration, paraffin embedding, and sectioning. The section thickness ranged from 3 μm.

[0082] The mixed stationary solution was a mixture of paraformaldehyde and glutaraldehyde, with paraformaldehyde concentration ranging from 3 wt% and glutaraldehyde concentration ranging from 0.5 wt%.

[0083] S3, First round of nano-gold labeling staining: Perform the first round of staining by incubating the tissue sample with the first staining agent at a temperature of 22℃ for 15 min;

[0084] During the incubation process, low-frequency ultrasound assistance is applied, with an ultrasound frequency range of 20kHz and a power density range of 0.5W / cm².

[0085] The first staining agent contains a specific antibody against the first target molecule, a secondary antibody labeled with gold nanoparticles, and a horseradish peroxidase substrate system. The gold nanoparticles have a particle size range of 5 nm and a concentration of 0.01 mg / mL.

[0086] The horseradish peroxidase substrate system is an enhanced chemiluminescent substrate containing luminol and hydrogen peroxide, with luminol concentrations ranging from 0.5 mmol / L to 0.01 wt%.

[0087] S4. Electrochemical-assisted controllable decolorization: The first round of controllable decolorization is carried out by treating tissue samples with a composite decolorizing agent. During the treatment, the pH value is controlled at 6.2, the treatment time is 8 min, and the solution oxidation-reduction potential is -200mV.

[0088] The composite decolorizing agent contains dithiothreitol, an electron shuttle medium, and a buffer solution. The electron shuttle medium is a ruthenium tripyridine complex with a concentration range of 0.5 mmol / L. A weak electric field is applied during the decolorization process, with an electric field strength range of 0.5 V / cm.

[0089] S5. Second round of quantum dot labeling staining: Perform the second round of staining by incubating the destained tissue sample with the second staining agent at a temperature of 22℃ for 15 minutes.

[0090] The second staining agent contains a specific antibody against the second target molecule, a quantum dot-labeled secondary antibody, and a fluorescent dye. The quantum dot particle size ranges from 3 nm, and the emission wavelength is adjustable from 450 nm. The quantum dot-labeled secondary antibody is coupled to the antibody via a click chemistry reaction. The click chemistry reaction uses a combination of cyclooctyne and azide, and the reaction time ranges from 30 min.

[0091] S6. Dynamic monitoring of cyclic staining: Repeat steps S4 and S5, each time using a different staining agent to label different target molecules. The number of repetitions is up to 1, and the destaining efficiency is evaluated using a dynamic optical monitoring system after each cycle.

[0092] The dynamic optical monitoring system used includes a spectrometer and a CCD detector, with a monitoring wavelength range of 400 nm, and automatically adjusts the subsequent decolorization time based on the monitoring results, with an adjustment time range of 1 min.

[0093] S7. Optical Transparency Treatment: A mixed solution containing urea and glycerol is used as an optical transparentizing agent to perform optical transparency treatment on multiple stained tissue samples;

[0094] The urea concentration range was 20 wt%, the glycerol concentration range was 30 wt%, and the treatment time was 1 hour.

[0095] S8. Multimodal Imaging Reconstruction: First, multimodal imaging analysis is performed on the processed tissue sample to obtain fluorescence images, dark-field images, and confocal images; then, based on the obtained images, three-dimensional reconstruction is performed using an image fusion algorithm.

[0096] Example 3

[0097] This application provides a multiple histochemical staining method based on a controlled staining-destaining cycle, comprising the following steps:

[0098] S1. Water bath-assisted antigen retrieval: Water bath-assisted antigen retrieval: Using EDTA alkaline retrieval solution, tissue samples are treated with water bath-assisted antigen retrieval. The retrieval temperature range is 100℃, and the retrieval time range is 40min.

[0099] Among them, the tissue samples were formalin-fixed paraffin-embedded tissues;

[0100] S2. Composite pretreatment: Composite pretreatment was performed on the repaired tissue samples. First, the samples were fixed with a mixed fixative for 8 hours, followed by gradient dehydration, paraffin embedding, and sectioning. The section thickness ranged from 8 μm.

[0101] The mixed stationary solution is a mixture of paraformaldehyde and glutaraldehyde, with paraformaldehyde concentration ranging from 6 wt% and glutaraldehyde concentration ranging from 2 wt%.

[0102] S3, First round of nano-gold labeling staining: Perform the first round of staining by incubating the tissue sample with the first staining agent at a temperature of 35℃ for 50 minutes;

[0103] Among them, low-frequency ultrasound assistance is applied during the incubation process, with an ultrasound frequency range of 40kHz and a power density range of 2W / cm².

[0104] The first staining agent contains a specific antibody against the first target molecule, a secondary antibody labeled with gold nanoparticles, and a horseradish peroxidase substrate system. The gold nanoparticles have a particle size range of 20 nm and a concentration of 0.5 mg / mL.

[0105] The horseradish peroxidase substrate system is an enhanced chemiluminescent substrate containing luminol and hydrogen peroxide, with luminol concentrations ranging from 10 mmol / L and hydrogen peroxide concentrations ranging from 0.1 wt%.

[0106] S4. Electrochemical-assisted controllable decolorization: The first round of controllable decolorization is carried out by treating tissue samples with a composite decolorizing agent. During the treatment, the pH value is controlled at 7.8, the treatment time is 25 min, and the solution oxidation-reduction potential is +100 mV.

[0107] The composite decolorizing agent contains dithiothreitol, an electron shuttle medium, and a buffer solution. The electron shuttle medium is a ruthenium bipyridine complex with a concentration range of 3 mmol / L. A weak electric field is applied during the decolorization process, with an electric field strength range of 2 V / cm.

[0108] S5. Second round of quantum dot labeling staining: Perform the second round of staining by incubating the destained tissue sample with the second staining agent at a temperature of 35°C for 50 minutes.

[0109] The second staining agent contains a specific antibody against the second target molecule, a quantum dot-labeled secondary antibody, and a fluorescent dye. The quantum dot particle size ranges from 15 nm, and the emission wavelength is adjustable from 650 nm. The quantum dot-labeled secondary antibody is coupled to the antibody via a click chemistry reaction. The click chemistry reaction uses a combination of cyclooctyne and azide, and the reaction time ranges from 90 min.

[0110] S6. Dynamic monitoring of cyclic staining: Repeat steps S4 and S5, each time using a different staining agent to label different target molecules, with a repetition range of 4 times, and use a dynamic optical monitoring system to evaluate the destaining efficiency after each cycle.

[0111] The dynamic optical monitoring system used includes a spectrometer and a CCD detector, with a monitoring wavelength range of 700 nm, and automatically adjusts the subsequent decolorization time based on the monitoring results, with an adjustment time range of 10 min.

[0112] S7. Optical Transparency Treatment: A mixed solution containing urea and glycerol is used as an optical transparentizing agent to perform optical transparency treatment on multiple stained tissue samples;

[0113] The urea concentration range was 40 wt%, the glycerol concentration range was 50 wt%, and the treatment time was 6 h.

[0114] S8. Multimodal Imaging Reconstruction: First, multimodal imaging analysis is performed on the processed tissue sample to obtain fluorescence images, dark-field images, and confocal images; then, based on the obtained images, three-dimensional reconstruction is performed using an image fusion algorithm.

[0115] Comparative Example 1

[0116] The only difference between this comparative example and Example 1 is that in step S2, the mixed fixative is a single paraformaldehyde solution with a concentration range of 4.5 wt%, and does not contain glutaraldehyde.

[0117] Comparative Example 2

[0118] The only difference between this comparative example and Example 1 is that in step S4, the electron shuttle medium in the composite decolorizing agent is methylene blue with a concentration range of 1.75 mmol / L, replacing the original ruthenium bipyridine complex.

[0119] Comparative Example 3

[0120] The only difference between this comparative example and Example 1 is that in step S7, the optical transparent agent is replaced with a single 40wt% glycerol solution, and the urea component is removed.

[0121] Comparative Example 4

[0122] The only difference between this comparative example and Example 1 is that in step S3, the incubation process of the first round of nano-gold labeling staining is not assisted by low-frequency ultrasound, but is statically incubated at 28.5°C for 32.5 min.

[0123] Comparative Example 5

[0124] The only difference between this comparative example and Example 1 is that in step S4, the step of applying a weak electric field is removed during the decolorization process, and only the pH value of 7.0, the treatment time of 16.5 min, and the solution redox potential of -50 mV are controlled.

[0125] Comparative Example 6

[0126] The only difference between this comparative example and Example 1 is that in step S5, the quantum dot-labeled secondary antibody is replaced by conventional glutaraldehyde crosslinking coupling instead of click chemical reaction coupling, while the coupling time remains 60 min.

[0127] I. Specificity and Signal Intensity Detection Experiment of Multiplex Staining

[0128] Stained tissue samples prepared according to their respective methods in Examples 1-3 and Comparative Examples 1-6 were selected, with three parallel samples for each type to ensure that the samples came from the same source and were formalin-fixed paraffin-embedded tissues or frozen tissues of the same tissue type. The samples were uniformly detected using confocal laser scanning microscopy and dark-field microscopy, corresponding to the sample types in each example.

[0129] First, standardized imaging parameters were set for the characteristic signals of the first and second target molecules in each sample: the quantum dot excitation wavelength matched the quantum dot emission wavelengths of Examples 1, 2, and 3, and the corresponding proportions; the laser intensity was fixed at 20%; and the exposure time was 100ms. Gold nanoparticles were observed using dark-field imaging with a fixed light source intensity of 50%. Subsequently, signal images of the target molecules were acquired for each sample, and ImageJ software was used to analyze the images: Firstly, the overlap rate between the positive signals of the target molecules and specific structures of tissue cells was statistically analyzed using the following formula: The first step is to measure the specific localization accuracy, denoted as FI value. The second step is to measure the average fluorescence intensity of the quantum dot fluorescence signal and the average gray value of the dark field signal of the gold nanoparticles, averaging the values ​​of three fields of view for each sample. Finally, the specific localization accuracy, FI value, and GS value of all examples and comparative examples are compared.

[0130] II. Decolorization Controllability and Cyclic Dyeing Stability Testing Experiment

[0131] Samples prepared in Examples 1-3 and Comparative Examples 1-6 were used as the detection objects. Three parallel samples were prepared for each type of sample to ensure strict adherence to each step of the sample processing procedure. A UV-Vis spectrophotometer and a dynamic optical monitoring system were used for detection. First, immediately after the first round of decolorization, the absorbance values ​​of the samples at the characteristic wavelengths of the target molecules were collected using a spectrometer: the characteristic absorption peak of gold nanoparticles is around 520 nm, and the characteristic absorption peak of quantum dots matches their emission wavelength. Three measurements were taken for each sample, and the average value was used to calculate the decolorization efficiency. The calculation formula is as follows: Subsequently, all staining cycles were completed according to the corresponding number of cycles for each sample. The absorbance measurement was repeated after each staining-destaining cycle, and the destaining efficiency was recorded for each cycle. Simultaneously, images of the samples after each staining cycle were acquired using a CCD detector. ImageJ software was used to calculate the percentage of overlap between the staining signal of the subsequent cycle and the residual signal of the previous cycle, which was recorded as the cross-interference rate. Furthermore, for samples that had completed all cycles, the specific localization accuracy of the target molecule was detected using the same method as in the multiple staining specificity and signal intensity detection experiment, evaluating the retention of staining specificity after multiple cycles.

[0132] III. Transparency Effect and 3D Imaging Quality Detection Experiment

[0133] Samples prepared in Examples 1-3 and Comparative Examples 1-6 that underwent transparentization were used, with three parallel samples for each type. After transparentization, all samples were allowed to stand at room temperature for 30 minutes before testing. A transmittance meter and a multimodal imaging system were used for detection. First, the transparentized sample was placed flat in the detection optical path of the transmittance meter. The transmittance was measured every 20 nm within the visible light range of 400-700 nm. The calculation formula is as follows: Three measurements were taken at each wavelength, and the average value was recorded. The transmittance values ​​of each sample at the quantum dot emission wavelengths of Examples 1, 2, and 3 were recorded. Subsequently, a multimodal imaging system was used to perform continuous tomographic scanning of the samples, with uniform scanning parameters: a scan step size of 1 μm, a scan range covering the entire thickness of the sample, and tomographic images of fluorescence, dark field, and confocal fields were acquired. The tomographic images were imported into Amira 6.7 software, and 3D reconstruction was performed using the same image fusion algorithm and 3D modeling parameters. Finally, the quality of the 3D model was analyzed: first, the spatial positioning error of the target molecule was calculated by measuring the deviation between the target molecule's positioning coordinates and its actual position, using the tissue anatomy as a reference, and taking the average value of three different regions; second, the integrity of the 3D structure was calculated using the following formula: .

[0134] The results of multiple staining specificity and signal intensity detection are shown in Table 1.

[0135] Table 1:

[0136] Group Specific localization accuracy (%) Average fluorescence intensity of quantum dots (au) Average gray value (au) of gold nanoparticles Example 1 98.5±0.5 8850±320 205±8 Example 2 97.2±0.8 7520±285 188±10 Example 3 96.8±1.0 9230±350 218±9 Comparative Example 1 85.3±2.5 7010±400 165±15 Comparative Example 2 90.1±1.8 7350±370 142±12 Comparative Example 3 97.0±0.9 5800±300 195±9 Comparative Example 4 92.5±1.5 6550±350 158±11 Comparative Example 5 88.7±2.0 6950±390 135±14 Comparative Example 6 83.6±2.8 5120±450 190±10

[0137] The results of the decolorization controllability and cyclic staining stability tests are shown in Table 2.

[0138] Table 2:

[0139] Group Average decolorization efficiency (%) Cross-interference rate (%) Post-cycle specificity retention rate (%) Example 1 98.2±0.5 1.5±0.5 98.0±0.7 Example 2 96.5±0.8 2.2±0.7 96.8±0.9 Example 3 97.8±0.6 1.8±0.6 97.5±0.8 Comparative Example 1 90.3±2.0 8.5±1.5 84.1±2.2 Comparative Example 2 75.6±3.5 22.3±3.0 88.9±2.0 Comparative Example 3 97.9±0.6 1.7±0.6 96.9±0.9 Comparative Example 4 95.8±0.9 3.5±0.9 95.1±1.1 Comparative Example 5 65.4±4.0 35.7±4.5 86.3±2.5 Comparative Example 6 97.5±0.7 2.0±0.7 82.5±2.8

[0140] The results of the transparency effect and the 3D imaging quality test are shown in Table 3.

[0141] Table 3:

[0142] Group Average transmittance (%) Spatial positioning error (μm) 3D structural integrity (%) Example 1 92.5±1.5 0.8±0.2 96.8±1.0 Example 2 88.0±2.0 1.2±0.3 94.5±1.5 Example 3 90.2±1.8 1.0±0.3 95.8±1.2 Comparative Example 1 85.5±2.5 2.5±0.6 88.3±2.5 Comparative Example 2 91.8±1.6 0.9±0.2 96.5±1.1 Comparative Example 3 65.3±4.0 5.8±1.0 75.4±3.5 Comparative Example 4 91.9±1.6 1.1±0.3 95.9±1.3 Comparative Example 5 92.1±1.5 0.9±0.2 96.2±1.1 Comparative Example 6 92.3±1.5 1.0±0.3 96.0±1.2

[0143] As can be seen from Examples 1-3 and Comparative Example 1, and in conjunction with Tables 1 and 2, the use of a single paraformaldehyde fixative significantly affects the stability of the tissue structure, leading to a decrease in the accuracy of marker localization during subsequent staining. It also weakens the tissue's ability to maintain its structure after undergoing multiple staining and decolorization cycles. This demonstrates the synergistic effect of glutaraldehyde and paraformaldehyde in forming a stable cross-linking network.

[0144] As can be seen from Examples 1-3 and Comparative Example 2, and Table 2, after replacing the ruthenium-based electron shuttle medium with conventional organic dyes, the efficiency and thoroughness of the decolorization process are significantly insufficient, leading to increased signal interference during cyclic dyeing. This indicates that metal complexes with specific structures have irreplaceable electron transfer efficiency in electrochemical decolorization systems and are a key factor in achieving efficient and controllable decolorization.

[0145] Combining Examples 1-3 and Comparative Example 3 with Tables 1 and 3, it can be seen that using only glycerol without urea in the optical transparency process will seriously affect the light transmittance of the tissue, which will lead to signal attenuation and spatial positioning distortion when imaging deep structures. This indicates that urea and glycerol have an important synergistic effect in changing the refractive index of tissues, and the two work together to achieve the ideal optical transparency effect.

[0146] As can be seen from Examples 1-3 and Comparative Example 4, and Table 1, the penetration and binding efficiency of the staining reagent were significantly affected after the low-frequency ultrasound assistance was removed, as evidenced by an overall weakening of the signal intensity. This confirms that physical field assistance can effectively promote molecular diffusion and interfacial reactions, and is an important process condition to ensure labeling efficiency.

[0147] As can be seen from Examples 1-3 and Comparative Example 5, and Table 2, when there is no electric field driving during the decolorization process, even if the same chemical decolorizing agent composition is maintained, the decolorization efficiency will be greatly reduced and the residual signal will be significantly increased. This reveals the necessity of electric field assistance for activating the electron shuttle medium and realizing directional electron transfer to achieve efficient decolorization.

[0148] As can be seen from Examples 1-3 and Comparative Example 6, and Tables 1 and 2, using traditional conjugation methods instead of click chemistry to prepare labeled antibodies leads to a decrease in antibody conjugation efficiency and specificity. This deficiency in initial labeling quality is accumulated and amplified during cyclic staining, ultimately affecting the overall effect of multiple staining, highlighting the fundamental role of efficient bioconjugation technology in the entire method system.

[0149] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A multiple histochemical staining method based on a controlled staining-destaining cycle, characterized in that: Includes the following steps: S1. Water bath-assisted antigen retrieval: Tissue samples are treated with water bath-assisted antigen retrieval using EDTA alkaline retrieval solution. The retrieval temperature range is 95-100℃, and the retrieval time range is 20-40 min. S2. Composite pretreatment: Composite pretreatment is performed on the repaired tissue samples. First, the samples are fixed with a mixed fixative for 2-8 hours, followed by gradient dehydration, paraffin embedding, and sectioning. The section thickness ranges from 3 to 8 μm. S3, First round of nano-gold labeling staining: Perform the first round of staining by incubating the tissue sample with the first staining agent at a temperature of 22-35℃ for 15-50 minutes; S4. Electrochemical-assisted controlled decolorization: Perform the first round of controlled decolorization by treating tissue samples with a composite decolorizing agent. During the treatment, control the pH value to 6.2-7.8, the treatment time to 8-25 min, and the solution oxidation-reduction potential to -200 mV to +100 mV. S5. Second round of quantum dot labeling staining: Perform the second round of staining by incubating the destained tissue sample with the second staining agent at a temperature of 22-35℃ for 15-50 minutes. S6. Dynamic monitoring of cyclic staining: Repeat steps S4 and S5, each time using a different staining agent to label different target molecules. The number of repetitions ranges from 1 to 4 times, and the destaining efficiency is evaluated using a dynamic optical monitoring system after each cycle. S7. Optical Transparency Treatment: A mixed solution containing urea and glycerol is used as an optical transparentizing agent to perform optical transparency treatment on multiple stained tissue samples; S8. Multimodal Imaging Reconstruction: First, multimodal imaging analysis is performed on the processed tissue sample to obtain fluorescence images, dark-field images, and confocal images; Subsequently, based on the obtained images, an image fusion algorithm was used for 3D reconstruction.

2. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S1, the tissue sample is formalin-fixed paraffin-embedded tissue or frozen tissue.

3. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S2, the mixed fixative is a mixture of paraformaldehyde and glutaraldehyde, wherein the concentration of paraformaldehyde ranges from 3 to 6 wt% and the concentration of glutaraldehyde ranges from 0.5 to 2 wt%.

4. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S3, low-frequency ultrasound is applied during the incubation process, with an ultrasound frequency range of 20-40kHz and a power density range of 0.5-2W / cm².

5. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S3, the first staining agent comprises a specific antibody against the first target molecule, a secondary antibody labeled with gold nanoparticles, and a horseradish peroxidase substrate system, wherein the gold nanoparticles have a particle size range of 5-20 nm and a concentration of 0.01-0.5 mg / mL.

6. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 5, characterized in that: The horseradish peroxidase substrate system is an enhanced chemiluminescent substrate containing luminol and hydrogen peroxide, with luminol concentration ranging from 0.5 to 10 mmol / L and hydrogen peroxide concentration ranging from 0.01 to 0.1 wt%.

7. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S4, the composite decolorizing agent comprises dithiothreitol, an electron shuttle medium, and a buffer solution. The electron shuttle medium is a ruthenium bipyridine complex or a ruthenium terpyridine complex with a concentration range of 0.5-3 mmol / L. A weak electric field is applied during the decolorization process, with an electric field strength range of 0.5-2 V / cm.

8. The multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S5, the second staining agent comprises a specific antibody against the second target molecule, a quantum dot-labeled secondary antibody, and a fluorescent dye, wherein the quantum dot particle size ranges from 3 to 15 nm, the emission wavelength is tunable in the range of 450 to 650 nm, and the quantum dot-labeled secondary antibody is coupled to the antibody via a click chemistry reaction. The click chemistry reaction uses a combination of cyclooctyne and azide, and the reaction time ranges from 30 to 90 min.

9. A multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S6, the dynamic optical monitoring system includes a spectrometer and a CCD detector, with a monitoring wavelength range of 400-700nm, and automatically adjusts the subsequent decolorization time according to the monitoring results, with an adjustment time range of 1-10min.

10. A multiple histochemical staining method based on a controllable staining-decolorization cycle according to claim 1, characterized in that: In step S7, the urea concentration ranges from 20-40 wt%, the glycerol concentration ranges from 30-50 wt%, and the treatment time is 1-6 hours.

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