Method for performing MALDI mass spectrum imaging detection and HE staining on medical tissue slice

Abstract

The invention relates to a method for performing MALDI mass spectrum imaging detection and HE staining on a medical tissue slice. The method comprises the following steps: S1, performing matrix spraying on the medical tissue slice, and performing MALDI mass spectrum imaging detection; s2, cleaning the medical tissue slice subjected to MALDI mass spectrum imaging detection by using a cleaning agent; s3, carrying out HE staining on the cleaned medical tissue slice; the cleaning agent in the step S2 comprises ethanol gel and a mixed solvent composed of acetone and tetrahydrofurfuryl alcohol diethyl ether; the volume ratio of acetone to tetrahydrofurfuryl alcohol ether in the mixed solvent is (1.85-4): 1; the ethanol gel is prepared from the following raw materials: ethanol, acetonitrile, PEG-400 and an ammonium formate aqueous solution. The method for performing MALDI mass spectrum imaging detection and HE staining on the medical tissue slice can ensure that the tissue is complete and free of matrix interference during HE staining.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for MALDI mass spectrometry imaging and HE staining of medical tissue sections. Background Technology

[0002] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) is a high-throughput molecular imaging technique that combines mass spectrometry analysis with spatial location information. It can directly detect the spatial distribution of hundreds to thousands of molecules (such as proteins, peptides, lipids, metabolites, and drugs) in biological tissue sections without labeling, providing a powerful molecular-level visualization tool for biological, medical, and pharmaceutical research. The analysis of carbohydrates / glycoconjugates (especially acidic sugars) is of profound significance in biology because they are widely involved in almost all core processes of life, and their structural complexity and diversity make them key carriers of biological information. Accurate resolution using appropriate analytical tools (such as MALDI-TOF MS on a DHT matrix) is crucial for understanding life mechanisms, disease diagnosis, and treatment.

[0003] Hematoxyline eosin staining, or HE staining for short, is the most basic and commonly used staining method for tissue sections in the fields of biology and medicine (especially histology and pathology). Its core purpose is to use two dyes to create a clear color contrast between different structures in the tissue, facilitating the observation and analysis of the basic morphology and structure of the tissue under an optical microscope.

[0004] The combined analysis of spatial metabolomics and hematoxylin and eosin (HE) staining is of great significance in biomedical research, particularly demonstrating unique advantages in tumor microenvironment studies, disease mechanism analysis, and precision medicine diagnosis. Mass spectrometry imaging reveals the spatial distribution of metabolites, such as lipids, amino acids, and carbohydrates, reflecting the metabolic heterogeneity of the microenvironment. Combined with the tissue morphological information provided by HE, precise localization is achieved, not only verifying the association between metabolites and pathological features, but also enabling multidimensional analysis from morphology to function to molecule.

[0005] Using adjacent consecutive slices (near-side slices) for spatial metabolic imaging and HE staining is one of the most common strategies, but it has significant drawbacks such as spatial offset, data heterogeneity, sample waste, and technical errors, which may affect the reliability of conclusions, especially in high-precision studies. Using the same slide for mass spectrometry imaging and HE staining has significant advantages in terms of spatial consistency, data reliability, sample conservation, and analytical efficiency. Summary of the Invention

[0006] To address the coordination inaccuracies and mapping issues caused by using separate MALDI mass spectrometry imaging and HE staining with slides, this application proposes a medical tissue HE staining method for MALDI mass spectrometry imaging of pentose phosphate pathway metabolites.

[0007] In a first aspect of this application, a method for MALDI mass spectrometry imaging and HE staining of medical tissue sections is provided, comprising the following steps: S1: Matrix spraying and MALDI mass spectrometry imaging detection of medical tissue sections; S2: Clean the medical tissue sections after MALDI mass spectrometry imaging with a cleaning agent; S3: Perform HE staining on the cleaned medical tissue sections; The cleaning agent in step S2 comprises ethanol gel and a mixed solvent consisting of acetone and tetrahydrofurfuryl ether; the volume ratio of acetone to tetrahydrofurfuryl ether in the mixed solvent is (1.85-4):1; The raw materials for preparing the ethanol gel include: ethanol, acetonitrile, PEG-400, and ammonium formate aqueous solution.

[0008] By employing the above technical solution, this application provides a cleaning agent for cleaning medical tissue sections after MALDI mass spectrometry imaging. Mouse lung tissue samples, after MALDI mass spectrometry imaging and matrix spraying treatment, are attached to ITO slides, placed flat on the operating table, covered with ethanol gel, and allowed to stand for adsorption. Afterward, they are transferred to a mixed solvent of acetone and THFEE for immersion and cleaning. This thoroughly removes the DHT matrix while ensuring the integrity of the tissue sample. Subsequent HE staining allows for 100% complete mapping of the HE staining image onto the tissue, clearly displaying tissue boundaries and micro-region structures, and enabling micro-region matching with the mass spectrometry imaging results. This is likely because the mixed solvent of acetone and THFEE, on the one hand, enhances the adhesion between the tissue and the slide, ensuring the integrity of the tissue sample; on the other hand, it separates the DHT matrix from the sample. Acetone penetrates into the DHT matrix fissures, expanding structural defects through solvation, while THFEE may destroy the internal structure of the DHT matrix, thus thoroughly removing the DHT matrix and ensuring the integrity of the tissue sample. Acetone and THFEE have a synergistic effect in removing DHT matrix and ensuring the integrity of medical tissue sections.

[0009] When the volume ratio of acetone to THFEE in the mixed solvent of acetone and THFEE is (1.85-4):1, it can ensure the integrity of the tissue and the absence of matrix interference during HE staining.

[0010] Optionally, the volume ratio of PEG-400 and ammonium formate aqueous solution in the raw materials for preparing the ethanol gel is 1:(5-11).

[0011] By employing the above-described technical solution and controlling the dosage of PEG-400 and ammonium formate aqueous solution according to the aforementioned volume ratio, a stable and effective gel carrier can be provided, along with a microenvironment that inhibits acid hydrolysis and protects tissue moisture and structure. This, in turn, promotes the complete removal of the DHT matrix while preserving tissue integrity, ensuring successful HE staining.

[0012] Optionally, the volume ratio of the ethanol, acetonitrile, PEG-400, and ammonium formate aqueous solution is 75:15:(0.2-0.4):(2.1-2.2).

[0013] By employing the above technical solution, acetonitrile, due to its high polarity, targets the phenolic hydroxyl groups of DHT; PEG-400 locks in tissue moisture and restricts the penetration depth of acetonitrile through steric hindrance, preventing cell structure damage; and ammonium formate aqueous solution forms a buffer system, inhibiting acid hydrolysis. These three components achieve synergistic effects through dynamic molecular equilibrium, enhancing the removal of the DHT matrix and maintaining the integrity of the tissue sample.

[0014] Optionally, the concentration of the ammonium formate aqueous solution is 4-6 mM.

[0015] Optionally, the method for preparing the ethanol gel includes: Step 1): Mix ethanol, acetonitrile, PEG-400 and ammonium formate aqueous solution to obtain the base solution; Step 2): The base solution is gelled to obtain the ethanol gel.

[0016] Optionally, in step 2), the gelation treatment of the base solution includes: dissolving gelatin in water to obtain a gelation solution, mixing the base solution with the gelation solution, stirring evenly, and allowing it to stand until gelation occurs to obtain the ethanol gel.

[0017] Optionally, the medical tissue section is a mouse lung tissue sample section; the MALDI mass spectrometry imaging detection uses a DHT matrix spray, and the MALDI mass spectrometry imaging detection includes the detection of pentose phosphate pathway metabolites in the medical tissue section.

[0018] By employing the above-mentioned technical solution, DHT matrix spraying was applied to medical tissue sections. Compared with DHB and SA spraying, DHT more effectively promoted the detection of carbohydrates, increasing the detection quantity by 47%. This may be because DHT itself is acidic (with two phenolic hydroxyl groups), generating an acidic environment during laser desorption / ionization, and more readily co-crystallizing with similar miscible compounds (such as carbohydrates, phosphate groups, sialic acid, etc.). Therefore, DHT matrix spraying is necessary to detect more carbohydrates.

[0019] Optionally, in step S2, the cleaning includes: covering the medical tissue slices after MALDI mass spectrometry imaging on the ITO slide with the ethanol gel, letting it stand, transferring it to a mixed solvent composed of acetone and THFEE for immersion, and then removing it.

[0020] Optionally, the soaking time is 3-5 minutes.

[0021] In a second aspect of this application, this application provides a cleaning agent for cleaning medical tissue slides after MALDI mass spectrometry imaging, characterized in that it comprises: an ethanol gel and a mixed solvent composed of acetone and tetrahydrofurfuryl ether, wherein the volume ratio of acetone to tetrahydrofurfuryl ether in the mixed solvent is (1.85-4):1; the raw materials for preparing the ethanol gel include: ethanol, acetonitrile, PEG-400, and ammonium formate aqueous solution.

[0022] Optionally, the volume ratio of PEG-400 and ammonium formate aqueous solution in the raw materials for preparing the ethanol gel is 1:(5-11).

[0023] Optionally, the volume ratio of the ethanol, acetonitrile, PEG-400, and ammonium formate aqueous solution is 75:15:(0.2-0.4):(2.1-2.2).

[0024] Optionally, the concentration of the ammonium formate aqueous solution is 4-6 mM.

[0025] Optionally, the method for preparing the ethanol gel includes: Step 1): Mix ethanol, acetonitrile, PEG-400 and ammonium formate aqueous solution to obtain the base solution; Step 2): The base solution is gelled to obtain the ethanol gel.

[0026] Optionally, in step 2), the gelation treatment of the base solution includes: dissolving gelatin in water to obtain a gelation solution, mixing the base solution with the gelation solution, stirring evenly, and allowing it to stand until gelation occurs to obtain the ethanol gel.

[0027] In a third aspect, this application provides the use of the cleaning agent described above for cleaning medical tissue slides after MALDI mass spectrometry imaging in cleaning medical tissue slides after MALDI mass spectrometry imaging.

[0028] Optionally, the medical tissue section is a mouse lung tissue sample section; the MALDI mass spectrometry imaging detection uses a DHT matrix spray, and the MALDI mass spectrometry imaging detection includes the detection of pentose phosphate pathway metabolites in the medical tissue section.

[0029] In summary, the present invention has at least one of the following beneficial technical effects: 1. This application provides a method for MALDI mass spectrometry imaging and HE staining of medical tissue sections. The MALDI mass spectrometry imaging uses a DHT matrix spray, which can be used to detect various pentose phosphate pathway metabolites. Compared with popular matrices such as DHB and SA, DHT is more effective in promoting the detection of carbohydrates, increasing the detection quantity by up to 47%. After MALDI mass spectrometry imaging, the sample is treated with the aforementioned cleaning agent to thoroughly remove the DHT matrix and ensure the integrity of the tissue sample. Subsequent HE staining allows for 100% complete mapping of the HE staining image onto the tissue, clearly displaying tissue boundaries and micro-region structures, and enabling matching of tissue micro-regions with the mass spectrometry imaging results.

[0030] 2. This application provides a cleaning agent for cleaning medical tissue sections after MALDI mass spectrometry imaging of pentose phosphate pathway metabolites. Mouse lung tissue samples, after MALDI mass spectrometry imaging and treatment with DHT matrix, are attached to ITO slides, placed flat on the operating table, covered with ethanol gel, and allowed to stand for adsorption. Afterward, they are transferred to a mixed solvent of acetone and THFEE for immersion and cleaning. This thoroughly removes the DHT matrix while maintaining the integrity of the tissue sample. Subsequent HE staining allows for 100% complete mapping of the HE staining image onto the tissue, clearly displaying tissue boundaries and micro-region structures, and enabling matching with the mass spectrometry imaging results of tissue micro-regions. The mixed solvent of acetone and THFEE, on the one hand, enhances the adhesion between the tissue and the slide, ensuring the integrity of the tissue sample; on the other hand, it separates the DHT matrix from the sample. Acetone penetrates into the DHT matrix fissures, expanding structural defects through solvation, while THFEE may destroy the internal structure of the DHT matrix, thus thoroughly removing the DHT matrix and ensuring the integrity of the tissue sample. Acetone and THFEE have a synergistic effect in removing DHT matrix and ensuring the integrity of medical tissue sections. Attached Figure Description

[0031] Figure 1 The mouse lung tissue sections from Example 1 of this application were sprayed with DHT matrix, and an ion map was generated to detect certain carbohydrates (galactose, trehalose, xylitol, glucose, glucose-6-phosphate, rhamnose, and threonic acid); among them, Figure 1 (A) Ion map showing the detection of galactose by spraying DHT matrix onto mouse lung tissue sections; Figure 1 (B) DHT matrix spraying of mouse lung tissue sections, and ion detection of trehalose; Figure 1 (C) DHT matrix spraying of mouse lung tissue sections, and the detection ion map of xylitol; Figure 1 (D) DHT matrix spraying of mouse lung tissue sections, and the detection ion map of glucose; Figure 1 (E) DHT matrix spraying of mouse lung tissue sections, and detection ion map of glucose-6-phosphate; Figure 1 (F) is a DHT matrix spraying of mouse lung tissue sections, showing the detection of rhamnose ions; Figure 1 (G) is a DHT matrix spraying of mouse lung tissue sections, and the detection ion map of threonine. Figure 2 A conventional HE-stained mouse lung tissue section image of adjacent sections in Example 2 provided in this application; Figure 3 The image is an HE staining image of the same mouse lung tissue section after MALDI mass spectrometry imaging detection, washing, and HE staining in Example 3 of this application. Figure 4 The HE staining image is obtained by washing and then HE staining the same mouse lung tissue section from Comparative Example 1 provided in this application after MALDI mass spectrometry imaging. Figure 5 The HE staining image is obtained by washing and then HE staining of the same mouse lung tissue section in Comparative Example 2 provided in this application after MALDI mass spectrometry imaging detection. Figure 6 The HE staining image is obtained by washing and then HE staining of the same mouse lung tissue section in Comparative Example 3 provided in this application after MALDI mass spectrometry imaging detection. Figure 7 The HE staining image is obtained by washing and then HE staining of the same mouse lung tissue section in Comparative Example 4 provided in this application after MALDI mass spectrometry imaging detection. Figure 8 The HE staining image is obtained by washing and then HE staining of the same mouse lung tissue section in Comparative Example 5 provided in this application after MALDI mass spectrometry imaging detection. Figure 9 The HE staining image is obtained by washing and then HE staining of the same mouse lung tissue section in Comparative Example 6 provided in this application after MALDI mass spectrometry imaging detection. Figure 10 The image shows a cleaned and then HE-stained lung tissue section of the same mouse in Comparative Example 7 provided in this application, obtained after MALDI mass spectrometry imaging. Detailed Implementation

[0032] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] This application proposes a method for detecting pentose phosphate pathway metabolites in medical tissue sections using MALDI mass spectrometry and HE staining.

[0034] Example 1 Mouse lung tissue samples were sprayed with DHT, DHB, and SA matrices, and then subjected to MALDI mass spectrometry imaging. The detection results of metabolites from the pentose phosphate pathway are shown in Table 1.

[0035] Table 1 As shown in Table 1, when mouse lung tissue samples were sprayed with DHT matrix, compared with DHB and SA spraying, DHT was more effective in promoting the detection of carbohydrates, and the detection quantity could be increased by 47%. Figure 1 The mouse lung tissue sections from Example 1 of this application were sprayed with DHT matrix, and the detection ion map of certain carbohydrates (galactose, trehalose, xylitol, glucose, glucose-6-phosphate, rhamnose, and threonic acid) was obtained by... Figure 1 It has been found that spraying mouse lung tissue samples with a DHT matrix can detect galactose, trehalose, xylitol, glucose, glucose-6-phosphate, rhamnose, and threonic acid. This is likely because DHT itself is acidic (with two phenolic hydroxyl groups), creating an acidic environment during laser desorption / ionization, and it more readily co-crystallizes with similar miscible compounds (such as sugars, phosphate groups, and sialic acid). Therefore, DHT matrix spraying is necessary to detect more sugars. However, it is difficult to ensure both thorough cleaning and preservation of the integrity of medical tissue sections during washing with DHT-matrixed samples.

[0036] Example 2 Two adjacent sections of mouse lung tissue were taken. One section was sprayed with DHT matrix and then used for MALDI mass spectrometry imaging; the other section was used for HE staining. Figure 2It is known that when using adjacent consecutive slices for spatial metabolic mass spectrometry imaging and HE staining, the HE staining of two adjacent slices can clearly show that there are significant differences at the edges of the sample (such as the surrounding contours and the central cavity). Even adjacent slices cannot achieve a reasonable fit. Therefore, adjacent slices often cause more significant errors when analyzing fine structures or high spatial resolution.

[0037] Based on Example 1, DHT matrix spraying on mouse lung tissue samples can more effectively promote the detection of carbohydrates, with a 47% increase in detection compared to DHB and SA spraying. Example 2 uses adjacent consecutive slices for spatial metabolic mass spectrometry imaging and HE staining. HE staining of adjacent slices can clearly show significant differences at the sample edges (regions with obvious differences in the surrounding contours and central cavities). Even adjacent slices cannot achieve a reasonable fit. Therefore, adjacent slices often lead to more significant errors when analyzing fine structures or high spatial resolution. In this invention, the same slice is sprayed with DHT matrix and then used for MALDI mass spectrometry imaging. The slices after mass spectrometry imaging are processed and then directly used for HE staining.

[0038] In the following examples, the same mouse lung tissue sections were sprayed with DHT matrix and then used for MALDI mass spectrometry imaging. The sections after mass spectrometry imaging were processed and then directly used for HE staining.

[0039] Preparation Example 1 The preparation steps for ethanol gel are as follows: Prepare the base solution: a1: Add 15 mL of acetonitrile to 75 mL of ethanol solution; a2: Add 0.3 mL of PEG-400; a3: Prepare 2.1 mL of 5 mM ammonium formate aqueous solution and add it, mix well to obtain the basic solution; Gelation treatment: b1: Dissolve 3 mL of gelatin in 4.6 mL of deionized water and heat until completely dissolved to obtain a gelled solution; b2: Mix the prepared base solution with the gelation solution and stir until homogeneous; b3: Let stand until gelation occurs to obtain ethanol gel.

[0040] Preparation Example 2 Preparation Example 2 provides an ethanol gel. The difference between Preparation Example 2 and Preparation Example 1 is that, in the preparation of the base solution, the total volume of PEG-400 and ammonium formate aqueous solution remains unchanged, and the volume ratio of PEG-400 and ammonium formate aqueous solution is 0.2:2.2.

[0041] Specifically, in the preparation of the base solution, step a2 is prepared differently, namely: adding 0.2 mL of PEG-400; step a3 is prepared differently, namely: preparing 2.2 mL of 5 mM ammonium formate aqueous solution and adding it, mixing evenly to obtain the base solution.

[0042] Preparation Example 3 Preparation Example 3 provides an ethanol gel. The difference between Preparation Example 3 and Preparation Example 1 is that, in the preparation of the base solution, the total volume of PEG-400 and ammonium formate aqueous solution in the ethanol gel remains unchanged, and the volume ratio of PEG-400 and ammonium formate aqueous solution is 0.4:2.0.

[0043] Specifically, step a2 is prepared differently, namely: adding 0.4 mL of PEG-400; step a3 is prepared differently, namely: preparing 2.0 mL of 5 mM ammonium formate aqueous solution and adding it, mixing evenly to obtain the basic solution.

[0044] Comparative preparation example 1: Comparative Preparation Example 1 provides an ethanol gel. The difference between Comparative Preparation Example 1 and Preparation Example 1 is that, in the preparation of the base solution, an equal volume of ammonium formate aqueous solution was replaced with PEG-400.

[0045] Specifically, step a3 is prepared differently, namely: 2.4 mL of PEG-400 is added and mixed evenly to obtain the basic solution.

[0046] Comparative preparation example 2: Comparative Preparation Example 2 provides an ethanol-blended solution. The difference between Comparative Preparation Example 2 and Preparation Example 1 is that the base solution is not subjected to gelation treatment after preparation. The preparation method of the ethanol compound solution is as follows: a1: Add 15 mL of acetonitrile to 75 mL of ethanol solution; a2: Add 0.3 mL of PEG-400; a3: Prepare 2.1 mL of 5 mM ammonium formate aqueous solution and add it, mix well to obtain the basic solution; a4: Mix the prepared base solution with 7.6 mL of deionized water and stir until homogeneous; a5: Let stand to obtain an ethanol compound solution.

[0047] Example 3 A method for MALDI mass spectrometry imaging and HE staining of pentose phosphate pathway metabolites in mouse lung tissue samples includes the following steps: S1: Mouse lung tissue samples were sliced ​​and sprayed with DHT matrix and subjected to MALDI mass spectrometry imaging to detect pentose phosphate pathway metabolites. The types of pentose phosphate pathway metabolites detected are shown in Table 1. S2: Place the mouse lung tissue sample slices (which have been treated with DHT matrix and attached to ITO slides) after MALDI mass spectrometry imaging flat on the operating table. Cover the tissue surface of the mouse lung tissue sample slices with 20 μL of ethanol gel prepared by the method in Preparation Example 1 and let it stand for 5 min. Then, transfer it to 25 mL of a pre-cooled mixed solvent of acetone and THFEE that has been treated with nitrogen to remove oxygen and soak it for 4 min. Remove it. The whole process is carried out in an ice bath at 4 °C. The volume ratio of acetone to THFEE in the mixed solvent is 7:3.

[0048] S3: After removal, hematoxylin and eosin (HE) staining is performed.

[0049] Figure 3 HE staining image of a section of mouse lung tissue sample.

[0050] Example 4 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue sample sections by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that the ethanol gel used in step S2 is different; the ethanol gel is the ethanol gel prepared by the method in Preparation Example 2.

[0051] Example 5 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue sample sections by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that the ethanol gel used in step S2 is different; the ethanol gel is the ethanol gel prepared by the method in Example 3.

[0052] Example 6 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue samples by MALDI mass spectrometry imaging and HE staining differs from Example 3 in that the mixed solvent of acetone and THFEE used in step S2 is different, and the volume ratio of acetone to THFEE in the mixed solvent is 8:2.

[0053] Example 7 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue samples by MALDI mass spectrometry imaging and HE staining differs from Example 3 in that the mixed solvent of acetone and THFEE used in step S2 is different, and the volume ratio of acetone to THFEE in the mixed solvent is 6.5:3.5.

[0054] The HE staining results of Examples 4-7 are similar to those of Example 3.

[0055] Comparative Example 1 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue sample sections by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that the ethanol gel used in step S2 is different; the ethanol gel is the ethanol gel prepared by the method of Comparative Preparation Example 1.

[0056] Figure 4 HE staining image of a section of mouse lung tissue sample.

[0057] Comparative Example 2 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue sample sections by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that, in step S2, an equal volume of the mixed solvent composed of acetone and THFEE is replaced with acetone.

[0058] Figure 5 HE staining image of a section of mouse lung tissue sample.

[0059] Comparative Example 3 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue sample sections by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that, in step S2, an equal volume of the mixed solvent composed of acetone and THFEE is replaced with THFEE.

[0060] Figure 6 HE staining image of a section of mouse lung tissue sample.

[0061] Comparative Example 4 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue samples using MALDI mass spectrometry and HE staining differs from Example 3 in that step S2 is different. In Comparative Example 4, step S2 involves placing a mouse lung tissue sample (treated with DHT matrix and attached to an ITO slide) after MALDI mass spectrometry imaging flat on the operating table, covering the tissue surface with 20 μL of ethanol gel prepared by the method in Preparation Example 1, and allowing it to stand for 5 min. Then, it is transferred to 25 mL of pre-cooled acetone treated with nitrogen for oxygen removal and soaked for 2 min. After soaking, it is transferred to 25 mL of pre-cooled THFEE treated with nitrogen for oxygen removal and soaked for 2 min. The entire process is carried out in an ice bath at 4°C.

[0062] Figure 7 HE staining image of a section of mouse lung tissue sample.

[0063] Comparative Example 5 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue samples using MALDI mass spectrometry and HE staining differs from Example 3 in that step S2 is different. In Comparative Example 5, step S2 involves placing a mouse lung tissue sample (treated with DHT matrix and attached to an ITO slide) after MALDI mass spectrometry imaging flat on the operating table, covering the tissue surface with 20 μL of ethanol gel prepared by the method in Preparation Example 1, and allowing it to stand for 5 min. Then, it is transferred to 25 mL of pre-cooled THFEE treated with nitrogen for oxygen removal and immersed for 2 min. After removal, it is transferred to 25 mL of pre-cooled acetone treated with nitrogen for oxygen removal and immersed for 2 min. The entire process is carried out in an ice bath at 4°C.

[0064] Figure 8 HE staining image of a section of mouse lung tissue sample.

[0065] Comparative Example 6 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue samples by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that the ethanol gel used in step S2 is different; the ethanol gel prepared by the method of Preparation Example 1 is replaced by an equal volume of the ethanol compound solution prepared by the method of Comparative Preparation Example 2.

[0066] Figure 9 HE staining image of a section of mouse lung tissue sample.

[0067] Comparative Example 7 A method for detecting pentose phosphate pathway metabolites in mouse lung tissue sample sections by MALDI mass spectrometry imaging and HE staining, differs from Example 3 in that, in step S2, a mixed solvent composed of ethanol and THFEE is used, with a volume ratio of 7:3.

[0068] Specifically, mouse lung tissue samples (treated with DHT matrix and attached to ITO slides) after MALDI mass spectrometry imaging were placed flat on the operating table. 20 μL of ethanol gel prepared according to the method in Example 1 was used to cover the tissue surface of the mouse lung tissue samples after MALDI mass spectrometry imaging and allowed to stand for 5 min. Then, the samples were transferred to 25 mL of a pre-cooled mixed solvent of ethanol and THFEE that had been treated with nitrogen to remove oxygen and immersed for 4 min. The entire process was carried out in an ice bath at 4 °C. The volume ratio of ethanol to THFEE in the mixed solvent was 7:3.

[0069] Figure 10 HE staining image of a section of mouse lung tissue sample.

[0070] Results Analysis and Summary Combining Example 3 and Figure 3 As can be seen, this invention uses the same mouse lung tissue slices for both mass spectrometry imaging and HE staining, employing the same tissue slide, thus achieving perfect alignment. Comparing the results before and after HE staining, it can be observed that even cavities in the lung tissue can be precisely identified. From... Figure 3 The staining effect shows that the DHT matrix can be cleaned thoroughly and maintain the integrity of the tissue.

[0071] In Examples 4-5, the volume ratio of PEG-400 and ammonium formate aqueous solution in the ethanol gel was adjusted, but all were within a suitable range. Therefore, the mass spectrometry imaging and HE staining effects of the same slice were almost the same as those in Example 3.

[0072] In Examples 6-7, the volume ratio of acetone to THFEE in the mixed solvent composed of acetone and THFEE was adjusted. Similarly, all adjustments were within a suitable range, so the mass spectrometry imaging and HE staining effects of the same slice were almost the same as in Example 3.

[0073] Comparison of Comparative Example 1 and Example 3 Figure 3 and Figure 4 It is known that replacing an equal volume of ammonium formate aqueous solution in ethanol gel with PEG-400 leads to an increase in DHT residual rate, resulting in matrix crystallization artifacts in the HE staining background and blurred tissue boundaries.

[0074] Comparison of Comparative Example 2, Comparative Example 3 and Example 3 Figure 3 and Figure 5 , Figure 6 It is known that using acetone or THFEE solution alone can lead to DHT matrix residue, which interferes with HE staining results.

[0075] Comparison of Example 4 and Example 3 Figure 3 and Figure 7 It can be seen that replacing the soaking in a mixed solvent of acetone and THFEE with soaking in acetone first and then THFEE resulted in severe tissue rupture and spatial mismatch between HE staining and mass spectrometry imaging.

[0076] Comparison of Comparative Example 5 and Example 3 Figure 3 and Figure 8 It can be seen that replacing the soaking in a mixed solvent of acetone and THFEE with soaking in THFEE solution first and then acetone solution resulted in severe tissue rupture.

[0077] Comparison of Comparative Example 6 and Example 3 Figure 3 and Figure 9 It can be seen that when the ethanol compound solution prepared in Comparative Preparation Example 2 is used instead of ethanol gel, the cell membrane structure is damaged after the surface water evaporates, which in turn affects the integrity of the tissue.

[0078] Comparison of Comparative Example 7 and Example 3 Figure 3 and Figure 10 It is known that when the mixed solvent of acetone and THFEE is replaced with a mixed solvent of ethanol and THFEE for washing, DHT matrix remains on the tissue surface, resulting in poor HE staining effect.

[0079] The above are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the products, methods and principles of this application should be covered within the scope of protection of this application.

Claims

1. A method for MALDI mass spectrometry imaging and HE staining of medical tissue sections, characterized in that, Includes the following steps: S1: Matrix spraying and MALDI mass spectrometry imaging detection of medical tissue sections; S2: Clean the medical tissue sections after MALDI mass spectrometry imaging with a cleaning agent; S3: Perform HE staining on the cleaned medical tissue sections; The cleaning agent in step S2 comprises ethanol gel and a mixed solvent consisting of acetone and tetrahydrofurfuryl ether; the volume ratio of acetone to tetrahydrofurfuryl ether in the mixed solvent is (1.85-4):1; The raw materials for preparing the ethanol gel include: ethanol, acetonitrile, PEG-400, and ammonium formate aqueous solution.

2. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 1, characterized in that, The volume ratio of PEG-400 and ammonium formate aqueous solution in the raw materials for preparing the ethanol gel is 1:(5-11).

3. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 1, characterized in that, The volume ratio of the aqueous solution of ethanol, acetonitrile, PEG-400, and ammonium formate is 75:15:(0.2-0.4):(2.1-2.2).

4. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 1, characterized in that, The concentration of the ammonium formate aqueous solution is 4-6 mM.

5. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 1, characterized in that, The method for preparing the ethanol gel includes: Step 1): Mix ethanol, acetonitrile, PEG-400 and ammonium formate aqueous solution to obtain the base solution; Step 2): The base solution is gelled to obtain the ethanol gel.

6. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 5, characterized in that, In step 2), the gelation treatment of the base solution includes: dissolving gelatin in water to obtain a gelation solution, mixing the base solution with the gelation solution, stirring evenly, and allowing it to stand until gelation occurs to obtain the ethanol gel.

7. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 1, characterized in that, The medical tissue section is a sample section of mouse lung tissue; the MALDI mass spectrometry imaging detection uses DHT matrix spraying, and the MALDI mass spectrometry imaging detection includes the detection of pentose phosphate pathway metabolites in the medical tissue section.

8. The method for MALDI mass spectrometry imaging and HE staining of medical tissue sections according to claim 1, characterized in that, Step S2 cleaning includes: Medical tissue sections subjected to MALDI mass spectrometry imaging on an ITO slide are covered with the ethanol gel, left to stand, then transferred to a mixed solvent of acetone and tetrahydrofurfuryl ether for immersion, and then removed.

9. A cleaning agent for cleaning medical tissue sections after MALDI mass spectrometry imaging, characterized in that, include: The ethanol gel is prepared from a mixed solvent consisting of acetone and tetrahydrofurfuryl ether, wherein the volume ratio of acetone to tetrahydrofurfuryl ether in the mixed solvent is (1.85-4):1; the raw materials for preparing the ethanol gel include ethanol, acetonitrile, PEG-400, and ammonium formate aqueous solution.

10. The use of the cleaning agent of claim 9 for cleaning medical tissue sections after MALDI mass spectrometry imaging in cleaning medical tissue sections after MALDI mass spectrometry imaging.

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