A method for processing a tissue section based on a contact prevention spacer

Abstract

This application discloses a method for processing tissue sections based on an anti-contact pad. The anti-contact pad has a window that extends through the anti-contact pad along its thickness. The processing method includes the following steps: preparing a glass slide and a tissue section, with the tissue section attached to the surface of the glass slide, and performing pre-treatment on the tissue section; preparing the anti-contact pad by placing it on the glass slide, ensuring that the tissue section or the area to be tested is completely within the window; adding a mounting medium or imaging medium into the window; preparing a coverslip by covering the anti-contact pad, so that the glass slide, anti-contact pad, and coverslip cooperate to form a spacer cavity to obtain a mounting assembly; acquiring a microscopic image of the mounting assembly; after image acquisition, placing the mounting assembly in a liquid environment; and after the physical properties of the mounting medium or imaging medium in the spacer cavity change, removing the coverslip and performing subsequent processing on the tissue section.

Description

Technical Field

[0001] This application relates to the field of biological tissue section processing and microscopic imaging technology, and in particular to a tissue section processing method based on an anti-contact pad. Background Technology

[0002] Immunohistochemical staining, immunofluorescence staining, and multiplex serial staining all require in-situ detection of biomarkers such as proteins on tissue sections. For formalin-fixed paraffin-embedded tissue sections, the tissue undergoes repeated temperature changes, buffer washing, organic solvent treatment, and mechanical disturbance during steps such as dewaxing, hydration, antigen retrieval, blocking, antibody incubation, staining, mounting, scanning, and destaining or inactivation. Especially in multiple rounds of serial staining, the same section needs to be repeatedly added to the solution, covered with coverslips, imaged, and the coverslips removed. The integrity and spatial stability of the tissue directly affect subsequent biomarker detection, image registration, and single-cell spatial analysis.

[0003] The standard procedure involves applying mounting medium or reaction solution to the tissue section and then directly covering it with a coverslip. Since the coverslip is in direct contact with the tissue surface, the weight of the coverslip itself, external forces during the procedure, liquid surface tension, coverslip sliding, and friction during removal can all cause localized pressure, detachment, or scraping of the tissue. These problems are more pronounced in samples with high heterogeneity, such as liver cancer, with numerous necrotic or fibrotic areas, and large section sizes.

[0004] Existing borders, sealing gaskets, or small reaction chambers are mainly used to restrict liquid flow, reduce evaporation, or create local reaction spaces. However, in multi-round continuous staining, if mounting media such as glycerol gelatin are used for bright-field imaging, the mounting media adhere to the coverslip, tissue section, and slide. Direct dry peeling or vertical prying of the coverslip can easily cause local tissue scraping, detachment, or displacement. While existing hydrophobic rings, open incubation boxes, or commercially available small reaction chambers can limit the diffusion of the reaction solution or maintain a humid environment, they do not provide a simple, low-cost solution compatible with conventional microscopy for the mechanical damage, localized compression, and unstable registration issues caused by direct contact between the coverslip and tissue in multi-round continuous staining. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a tissue sectioning method based on anti-contact pads, which can reduce tissue area loss during multiple rounds of continuous processing, mounting, image acquisition, and coverslip removal, and improve tissue morphology preservation, tissue area retention rate, and registration stability of subsequent images.

[0006] According to an embodiment of this application, a tissue sectioning method based on an anti-contact pad is provided. The anti-contact pad has a window that extends through the anti-contact pad along its thickness direction. The tissue sectioning method based on the anti-contact pad includes the following steps: Prepare glass slides and tissue sections, with the tissue sections attached to the surface of the glass slides, and perform pre-treatment on the tissue sections for detection; Prepare the anti-contact pad, place the anti-contact pad on the glass slide, and ensure that the tissue section or the area to be tested is completely within the window; Add a sealing material or imaging medium into the window; Prepare a cover glass and cover it over the anti-contact pad, so that the slide, the anti-contact pad and the cover glass cooperate to form a spacer cavity to obtain the sealing assembly; Microscopic images are acquired from the mounting assembly; After image acquisition is completed, the sealing assembly is placed in a liquid environment; After the physical properties of the mounting medium or the imaging medium in the spacer cavity have changed, the coverslip is removed, and the tissue section is then processed.

[0007] The tissue section processing method according to the embodiments of this application has at least the following beneficial effects: by setting an anti-contact pad around the tissue section to support the coverslip, a spacer cavity defined by the thickness of the pad is formed between the coverslip and the tissue section, avoiding direct contact or pressure of the coverslip on the tissue section; simultaneously, after image acquisition, the coverslip is removed after the mounting medium or imaging medium has softened, swelled, or loosened in a liquid environment, avoiding mechanical damage caused by dry peeling or vertical prying of the coverslip. The combination of the aforementioned anti-contact mounting and gentle coverslip removal in a liquid environment can reduce local pressure, scraping, detachment, and displacement of the tissue section caused by direct pressure, slippage, and friction of the coverslip during mounting, image acquisition, and coverslip removal, thereby improving the morphological preservation, tissue area retention rate, and subsequent image registration stability of the tissue section.

[0008] According to some embodiments of this application, the steps of placing the mounting assembly in a liquid environment and removing the coverslip after the mounting medium or imaging medium in the spacer cavity has softened, swollen, or loosened, and then performing subsequent processing on the tissue section, include: The cover glass assembly is immersed in a buffer solution and placed in a water bath at a set temperature, causing the cover glass or imaging medium in the spacer cavity to soften, swell, or loosen. Then, force is applied to the cover glass in the environment of the buffer solution to separate the cover glass from the anti-contact pad.

[0009] According to some embodiments of this application, in the step of adding a sealing material or imaging medium into the window, the amount of the sealing material or imaging medium added is determined based on the thickness of the anti-contact pad and the area of ​​the window, such that the sealing material or imaging medium substantially fills the spacer cavity.

[0010] According to some embodiments of this application, the mounting medium comprises glycerol gelatin, the buffer solution comprises phosphate buffer, and the water bath is set to a temperature range of 36.5°C-37.5°C.

[0011] According to some embodiments of this application, before the steps of preparing the anti-contact pad, placing the anti-contact pad on the glass slide, and ensuring that the tissue section or the area to be tested is completely within the window, the following steps are further included: Select the appropriate size of the anti-contact pad. The overall size and window size of the anti-contact pad are determined based on at least two of the following: the size of the tissue section or the size of the area to be tested, the size of the coverslip, the size of the slide, the thickness of the tissue section, the viscosity of the mounting medium, and the requirements of the focal plane of the microscopy scan.

[0012] According to some embodiments of this application, the distance between the edge of the tissue slice or the area to be detected and the inner edge of the window ranges from 0.5mm to 5mm, the distance d between the outer side of the anti-contact pad and the inner edge of the window ranges from 0.3mm to d to 8mm, and the thickness h of the anti-contact pad ranges from 20μm to h to 500μm.

[0013] According to some embodiments of this application, the distance d between the outer side of the anti-contact pad and the inner edge of the window ranges from 0.5mm to 5mm, and the thickness h of the anti-contact pad ranges from 50μm to 150μm.

[0014] According to some embodiments of this application, the step of preparing the anti-contact pad, placing the anti-contact pad on the glass slide, and ensuring that the tissue section or the area to be tested is completely within the window includes: An adhesive layer is provided on the surface of the anti-contact pad for bonding with the glass slide, and the anti-contact pad is bonded to the glass slide through the adhesive layer to fix the anti-contact pad.

[0015] According to some embodiments of this application, after the physical properties of the mounting medium or the imaging medium in the spacer cavity change, the coverslip is removed, and subsequent processing steps are performed on the tissue section, the following steps are further included: After the tissue section is processed for the next round of detection, the operations of adding the mounting medium or the imaging medium, covering the coverslip, image acquisition, and removing the coverslip are repeated. The above steps are repeated multiple times until the predetermined round of detection is completed.

[0016] According to some embodiments of this application, the pre-processing of the tissue section for detection includes: When the tissue section is a formalin-fixed paraffin-embedded tissue section, the pre-detection treatment includes dewaxing, hydration, and antigen retrieval; when the tissue section is a frozen tissue section, the pre-detection treatment includes fixation and washing.

[0017] The tissue section processing method according to the above embodiments of this application has at least the following beneficial effects: 1. Immersing the mounting assembly in buffer solution and placing it in a temperature-controlled water bath ensures uniform softening of the mounting medium rather than localized hardening residue, reducing shear and friction forces during cover plate removal. Using glycerol gelatin in combination with phosphate buffer solution and a 36.5℃-37.5℃ water bath effectively softens the mounting medium without damaging the tissue structure. 2. Selecting the window size, width, and thickness of the gasket according to the tissue section specifications and imaging requirements, and determining the amount of mounting medium used based on the gasket thickness and window area to fully fill the spacer cavity, ensures the formation of a continuous and uniform imaging medium layer, avoids air bubbles and uneven liquid layers, and meets the focusing requirements of brightfield or fluorescence scanning. 3. An adhesive layer is provided on the surface of the anti-contact gasket to adhere the cover plate, preventing cover plate slippage during image acquisition, maintaining a constant spacer cavity distance, improving focal plane stability and cross-cycle image registration accuracy. 4. Each round utilizes a combination of contact-resistant sealing and gentle liquid environment for cover removal, keeping mechanical damage per round at a low level and preventing it from accumulating and amplifying with each round. Experiments show that the cumulative area loss rate of large tissue sections after 5 rounds of continuous staining is still ≤0.64%, and this method is compatible with high-round continuous processing of large tissue sections and tissue microarrays.

[0018] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0019] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of the anti-contact pad according to Embodiment 1 of this application; Figure 2 This is a schematic diagram of the sealing assembly of Embodiment 1 of this application; Figure 3 This is a flowchart of the method for multi-round continuous staining of tissue sections in Embodiment 1 of this application; Figure 4This is a comparison image of the scanning results of staining and image acquisition performed using the processing method of this application in Example 2 and staining and image acquisition performed using conventional methods; Figure 5 These are representative scan images comparing five rounds of continuous staining of a large tissue section using the processing method of this application and conventional processing methods, respectively, in Example 3. Figure 6 This is a line graph comparing the cumulative area loss rate during five rounds of continuous staining of a large tissue section using the processing method of this application and conventional processing methods, respectively; Figure 7 This is a representative image of high-round continuous processing and image acquisition of a large tissue area using the processing method of this application in Example 4; Figure 8 This is a representative image of the tissue chip being processed and image acquired in high-round continuous processing using the processing method of this application in Example 4.

[0020] Reference numerals: 100, anti-contact pad; 110, window; 200, glass slide; 300, cover glass. Detailed Implementation

[0021] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0022] In the description of this application, it should be understood that if directional descriptions are involved, such as up, down, front, back, left, right, etc., indicating the directional or positional relationship based on the directional or positional relationship shown in the accompanying drawings, it is only for the convenience of describing this application and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0023] In the description of this application, if words such as several, greater than, less than, exceeding, above, below, or within appear, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the number itself, and "above," "below," and "within" are understood to include the number itself.

[0024] In the description of this application, the use of terms such as "first" and "second" is for the purpose of distinguishing technical features only, and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or the order of the technical features indicated.

[0025] In the description of this application, unless otherwise expressly defined, terms such as "setup," "installation," and "connection" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this application in conjunction with the specific content of the technical solution.

[0026] Example 1 Combination Figures 1-3 According to an embodiment of this application, a tissue sectioning method based on an anti-contact pad uses an anti-contact pad 100, which has a window 110 extending through the anti-contact pad 100 along its thickness direction. The tissue sectioning method based on the anti-contact pad includes the following steps: S1. Prepare glass slide 200 and tissue sections. The tissue sections are attached to the surface of glass slide 200 and pre-treatment is performed on the tissue sections for detection.

[0027] It should be noted that the processing method of this application can be applied to continuous in situ detection procedures of tissue sections in immunohistochemistry, immunofluorescence, nucleic acid in situ hybridization, protein in situ detection, or other procedures requiring repeated mounting and image acquisition. In these procedures, tissue sections are typically stained to label target molecules in each round of detection. Staining can be direct or indirect, such as using enzyme-labeled antibodies, fluorescently labeled antibodies, or labeled nucleic acid probes. After staining, the tissue sections are mounted to protect the signal and facilitate imaging. After image acquisition, the mounting medium and / or at least part of the signal can be removed to allow for the next round of staining and imaging of the same section. The above cycle can be repeated multiple times to achieve continuous in situ detection of multiple target molecules on the same tissue section.

[0028] The tissue sections used in this step include, but are not limited to, formalin-fixed paraffin-embedded tissue sections, frozen tissue sections, tissue microarrays, cell slides, or other biological samples that can adhere to the surface of a glass slide. In this embodiment, the thickness of the tissue sections is selected to be 2–10 micrometers, more preferably 3–5 micrometers.

[0029] In this embodiment, the pretreatment of tissue sections in this step is the pretreatment of tissue sections for staining. For example, for formalin-fixed paraffin-embedded tissue sections, the process may include baking, dewaxing, hydration, antigen retrieval, endogenous enzyme inactivation, and blocking; for frozen tissue sections, the process may include fixation, washing, and blocking. After completing staining, development, or other in-situ detection steps, the next step can be performed.

[0030] S2. Select an appropriate size of anti-contact pad 100. In this embodiment, the anti-contact pad 100 adopts a rectangular frame structure. The window 110 size and overall size of the anti-contact pad 100 are determined based on at least two of the following: the size of the tissue section or the area to be tested, the size of the coverslip 300, the size of the slide 200, the thickness of the tissue section, the viscosity of the mounting medium, and the focal plane requirements for microscopic scanning. The basic requirement is that the window 110 should completely surround the tissue section or the area to be tested, and the tissue section should not be covered or compressed by the anti-contact pad 100.

[0031] The anti-contact pad 100 is used to support the coverslip 300. The distance d between the outer surface of the anti-contact pad 100 and the inner edge of the window 110 should be selected to ensure stable support of the coverslip 300 without obstructing the area of ​​the tissue to be examined. The thickness h of the anti-contact pad 100 is used to limit the distance between the lower surface of the coverslip 300 and the surface of the tissue section.

[0032] According to some embodiments of this application, the distance between the edge of the tissue slice or the area to be detected and the edge of the window 110 ranges from 0.5mm to 5mm, the distance d between the outer side of the anti-contact pad 100 and the inner edge of the window 110 ranges from 0.3mm to d to 8mm, and the thickness h of the anti-contact pad 100 ranges from 20μm to h to 500μm.

[0033] According to some other embodiments of this application, the range of d is 0.5mm ≤ d ≤ 5mm, and the range of h is 50μm ≤ h ≤ 150μm. This thickness selection avoids direct contact between the coverslip 300 and the tissue section, while allowing the mounting medium or imaging medium to form a continuous and relatively uniform thin layer on the surface of the tissue section, and meeting the focusing requirements of bright-field or fluorescence scanning.

[0034] S3. Positioning and Installation of the Anti-Contact Pad. Prepare the anti-contact pad and place the anti-contact pad 100 on the glass slide 200 and around the tissue section, ensuring that the tissue section or the area to be tested is completely within the window 110, and avoiding the anti-contact pad covering or contacting the tissue section. For large tissue sections, the position of the anti-contact pad window 110 can be adjusted according to the morphology of the tissue's outer edge, so that the main tissue area is completely within the window 110. For tissue microarrays, the entire lattice area of ​​the tissue microarray can be placed within the window 110.

[0035] In some embodiments, the anti-contact pad is installed and positioned as follows: an adhesive layer is provided on the lower surface of the anti-contact pad for contact with the glass slide. The adhesive layer is made of an adhesive material that physically adheres to the glass. The anti-contact pad is bonded to the glass slide through the adhesive layer so that the anti-contact pad remains in a stable position.

[0036] It should be noted that in other embodiments, the shape, material, thickness, and fixing method of the anti-contact pad 100 can be replaced as appropriate.

[0037] S4. Add mounting medium or imaging medium into window 110. In this embodiment, glycerol gelatin is used as the mounting medium. After being added, the glycerol gelatin covers the surface of the tissue section and forms a continuous imaging medium layer after being covered by coverslip 300. Air bubbles should be avoided from entering the tissue area during sample addition. In other embodiments, glycerol gelatin can be replaced with other mounting medium or imaging medium that is compatible with tissue staining and image acquisition and can be softened, swollen, or removed by an aqueous medium.

[0038] S5. Prepare a coverslip. Place the coverslip 300 over the anti-contact pad 100, so that the slide, anti-contact pad, and coverslip cooperate to form a spacer cavity to obtain the sealing assembly. When covering the coverslip 300, it can be slowly lowered from one side to the other, allowing the glycerin gelatin to gradually spread, thereby reducing air bubble residue and uneven local liquid layer. In step S4, the amount of sealing material or imaging medium added is determined according to the thickness of the anti-contact pad 100 and the area of ​​the window 110 region, so that the sealing material or imaging medium basically fills the spacer cavity.

[0039] In some embodiments, the coverslip 300 is installed and fixed in such a way that the coverslip 300 can maintain a stable position through its own gravity, the surface tension of the liquid in the spacer cavity, the adaptation structure between the coverslip and the slide, and / or external limiting.

[0040] By setting an adhesive layer on the surface of the anti-contact pad to bond the cover glass 300, the cover glass 300 is prevented from slipping during image acquisition, the interval cavity distance is kept constant, and the focal plane stability and cross-cycle image registration accuracy are improved.

[0041] S6. The obtained mounting assembly is subjected to microscopic image acquisition, including bright-field scanning, fluorescence scanning, or other microscopic image acquisition. The mounting assembly includes tissue sections, a glass slide 200, a coverslip 300, and an anti-contact pad 100. Since the coverslip 300 is supported by the anti-contact pad 100, the tissue sections are not directly compressed by the coverslip 300 during image acquisition, which helps to reduce local tissue deformation, displacement, and imaging instability caused by compression.

[0042] S7. After image acquisition is complete, place the sealing assembly in a liquid environment.

[0043] S8. After the physical properties of the mounting medium or imaging medium in the spacer cavity change in the liquid environment, remove the coverslip 300 and perform subsequent processing on the tissue section.

[0044] Specifically, the sealing assembly is immersed in a buffer solution and placed in a water bath at a set temperature, causing the sealing material or imaging medium in the spacer cavity to soften, swell, or loosen. Then, force is applied to the coverslip in the buffer solution environment to separate the coverslip from the anti-contact pad.

[0045] According to some embodiments of this application, the buffer solution is phosphate buffer, and the water bath temperature is set within the range of 36.5℃-37.5℃. The mounting assembly is completely immersed in the phosphate buffer and placed in a water bath at 37℃ to gradually soften, swell, or loosen the glycerol gelatin under the coverslip 300. After the coverslip 300 and the anti-contact pad can slide relative to each other, the coverslip 300 is gently pried or pushed along the edge parallel to the coverlip in the liquid environment of the phosphate buffer to separate the coverslip 300 from the anti-contact pad 100 and the slide 200.

[0046] During the removal of the coverslip, avoid peeling it off dry, avoid prying it vertically, and avoid directly scraping the tissue area with the edges of the coverslip. After removing the coverslip, wash the tissue sections with phosphate-buffered saline (PBFS) or perform other subsequent treatments. In some other embodiments, PBFS can be replaced with other buffers compatible with the tissue sections and mounting medium; the 37°C water bath can also be adjusted to other suitable temperatures depending on the softening properties of the mounting medium.

[0047] Immersing the mounting assembly in buffer solution and placing it in a temperature-controlled water bath ensures uniform softening of the mounting medium, preventing localized hardening and residue, thus reducing shear and friction forces when removing the coverslip. Using glycerol gelatin in combination with phosphate buffer solution and a 36.5℃-37.5℃ water bath effectively softens the mounting medium without damaging tissue structures.

[0048] S9. After performing the pretreatment of the tissue section for the next round of testing, repeat steps S4-S8 to add the mounting medium or the imaging medium, cover with a coverslip, acquire the image, and remove the coverslip. Repeat the above steps multiple times until the predetermined round of testing is completed.

[0049] Each round utilizes a combination of contact-resistant sealing and gentle removal of the coverslip in a liquid environment to keep mechanical damage in a single round at a low level and prevent it from accumulating and amplifying with each round. Experiments show that the cumulative area loss rate of large tissue sections after 5 rounds of continuous staining is still ≤0.64%, and this method is compatible with high-round continuous processing of large tissue sections and tissue microarrays.

[0050] The tissue section processing method according to the embodiments of this application has at least the following beneficial effects: by setting an anti-contact pad around the tissue section to support the coverslip, a spacer cavity defined by the thickness of the pad is formed between the coverslip and the tissue section, avoiding direct contact or pressure of the coverslip on the tissue section; simultaneously, after image acquisition, the coverslip is removed after the mounting medium or imaging medium has softened, swelled, or loosened in a liquid environment, avoiding mechanical damage caused by dry peeling or vertical prying of the coverslip. The combination of the aforementioned anti-contact mounting and gentle coverslip removal in a liquid environment can reduce local pressure, scraping, detachment, and displacement of the tissue section caused by direct pressure, slippage, and friction of the coverslip during mounting, image acquisition, and coverslip removal, thereby improving the morphological preservation, tissue area retention rate, and subsequent image registration stability of the tissue section.

[0051] Example 2 Formalin-fixed paraffin-embedded tissue sections were prepared by baking, dewaxing, hydration, antigen retrieval, endogenous enzyme inactivation, blocking, and antibody incubation before staining. The control group underwent conventional staining and scanning with direct coverslips; the experimental group used the contact-avoidance mounting method S2-S5 described in Example 1 of this application, which involves placing a contact-avoidance pad around the tissue section, ensuring the tissue area is completely within the window of the pad, and avoiding the pad covering or compressing the tissue section. Glycerin gelatin was then added to the window as a mounting medium or imaging medium, and a coverslip was placed over it, supported by the contact-avoidance pad and not in direct contact with the tissue section.

[0052] After mounting, bright-field microscopy was performed on the tissue sections. After image acquisition, the slides were immersed in phosphate buffer and placed in a 37°C water bath to gradually soften or loosen the glycerol gelatin. Then, the coverslips were gently moved or pushed along the edge of the coverslip in a liquid environment. After removing the coverslips, they were washed or subjected to further processing.

[0053] refer to Figure 4 As shown in the image acquisition results, the contactless mounting method of this application allows for clear observation of positive staining signals in tissue sections, and the local cell morphology and staining distribution meet the requirements for image acquisition and subsequent quantitative analysis. Compared with conventional direct coverslip scanning images, the contactless mounting method of this application does not significantly affect the observation of positive signals and the identification of local structures within the tissue area. This result demonstrates that the processing method of this application is compatible with conventional tissue section staining, glycerol gelatin mounting, and microscopic image acquisition workflows.

[0054] Example 3 Large sections of formalin-fixed paraffin-embedded tissue were taken, and after pre-staining treatment, continuous staining and image acquisition were performed using the processing method of Example 1 of this application and conventional processing methods, respectively.

[0055] In the experimental group using contact-resistant mounting, contact-resistant pads were placed around the tissue sections, ensuring the main tissue area remained within the frame pad window. After each round of staining or development, glycerol gelatin was added to the frame pad window as a mounting medium or imaging medium, and a coverslip was placed over it, supported by the frame pad and not in direct contact with the tissue section. After image acquisition, the slide was immersed in phosphate buffer and placed in a 37°C water bath to gradually soften or loosen the glycerol gelatin. Subsequently, the coverslip was gently moved or pushed along its edge in the liquid environment to remove it. After removing the coverslip, the tissue sections were washed and prepared for the next round of processing.

[0056] In the control group using the standard mounting method, no anti-contact pad was used. After adding mounting medium or imaging medium to the surface of the tissue section, a coverslip was directly placed over it using the standard method, ensuring the coverslip covered the tissue section. After image acquisition, the coverslip was removed using the standard procedure, followed by washing and the next processing step.

[0057] In sequential staining, scan images of the same tissue section from different rounds are compared. (Reference) Figure 5 As shown, the results indicate that the tissue sections treated with the anti-contact mounting method maintained a relatively good overall tissue outline and high continuity throughout the first to fifth rounds of continuous staining. In contrast, the conventional mounting method resulted in visible local tissue defects or reduced tissue area in subsequent rounds. These results demonstrate that conventional mounting and removal methods are more likely to cause local tissue loss during multiple rounds of mounting, image acquisition, coverslip removal, and liquid processing. The processing method described in this application reduces the mechanical damage to tissue sections caused by direct pressure, slippage, or removal of coverslips.

[0058] To further quantitatively verify the above observations, this embodiment uses the tissue area in the first round of images as the baseline area, calculates the cumulative area loss rate in subsequent rounds, and compares the differences between the group using the contactless mounting method and the group using the conventional mounting method. The cumulative area loss rate is calculated as follows: the identifiable tissue area in the first round of scanned images is used as the baseline area, denoted as A1; the identifiable tissue area in the subsequent nth round of scanned images is used as An; the cumulative area loss rate in the nth round is calculated using the formula (A1 - An) / A1 × 100%. Each tissue slide is treated as an independent sample, and the cumulative area loss rate for rounds 2 to 5 is calculated separately. The calculation results are shown in the table below: Table 1. Effect of the processing method of this application on the cumulative area loss rate during multiple rounds of continuous staining of large tissue areas.

[0059] Note: The loss rate data in Table 1 are expressed as mean ± standard deviation. The difference in loss rates is the cumulative area loss rate of the conventional sealing method group minus the cumulative area loss rate of the contactless sealing method group. The P-value is calculated using a two-sided exact permutation test to compare the difference in cumulative area loss rates between the two groups in each round.

[0060] The results showed that in the second round, the cumulative area loss rate of both groups was low, with no significant difference; the rate was 0.26±0.21% for the contactless sealing method group and 0.34±0.39% for the conventional sealing method group, with a difference of 0.08% (P=0.714). From the third round onwards, the cumulative area loss rate of the conventional sealing method group increased significantly, while the contactless sealing method group remained at a low level. In the third round, the cumulative area loss rate of the contactless sealing method group was 0.50±0.33%, while that of the conventional sealing method group was 4.07±5.77%, with a difference of 3.57% and P=0.0238. In the fourth round, the rate was 0.53±0.33% for the contactless sealing method group and 5.87±5.89% for the conventional sealing method group, with a difference of 5.34% and P=0.00794. In the fifth round, the rate was 0.64±0.34% for the contactless sealing method group and 6.76±6.48% for the conventional sealing method group, with a difference of 6.12% and P=0.00794.

[0061] refer to Figure 6 As shown, the above data results are visually displayed in a comparison line chart. Figure 6 The left figure shows the conventional mounting method group, and the right figure shows the contactless mounting method group, with 5 samples in each group. The horizontal axis represents the number of consecutive staining rounds, with R1–R5 representing rounds 1 to 5, respectively; the vertical axis represents the cumulative area loss rate. The light gray line shows the trend of individual tissue section samples in different rounds, and the dark gray line and error bar represent the mean ± standard deviation within the group. Statistical analysis used a two-tailed exact permutation test; This indicates that P < 0.05. This indicates that P < 0.01.

[0062] According to Table 1 and Figure 6 The results showed that, compared with the conventional sealing method group, the contact-resistant sealing method group had a significantly lower cumulative area loss rate in the 3rd, 4th and 5th rounds.

[0063] The above results indicate that during multiple rounds of continuous staining, the tissue area loss in the conventional mounting method group accumulated with each round, and the fluctuations between samples were significantly larger. However, after adopting the contactless mounting and coverslip removal method of this application, the cumulative tissue area loss rate remained at a low level throughout the first to fifth rounds. Therefore, the combination of the contactless mounting method and the gentle liquid environment for coverslip removal in this application can reduce tissue area loss during mounting, image acquisition, and coverslip removal, thereby improving the morphological preservation of tissue sections and the stability of multi-round detection.

[0064] Example 4 Formalin-fixed paraffin-embedded tissue sections or tissue microarray sections were pretreated for staining and then continuously processed using the method described in Example 1 of this application. Specifically, a contact-proof frame spacer was placed around the tissue section to ensure that the main area of ​​the tissue section or the lattice area of ​​the tissue microarray was completely within the window of the frame spacer. After each round of staining or development, glycerol gelatin was added to the window of the frame spacer as a mounting medium or imaging medium, and a coverslip was placed over it. The coverslip was supported by the contact-proof frame spacer and did not directly contact or compress the tissue section.

[0065] After each round of image acquisition, the slide is immersed in phosphate buffer and placed in a 37°C water bath to gradually soften or loosen the glycerol gelatin. Then, the coverslip is gently moved or pushed along its edge in a liquid environment to remove it. After removing the coverslip, the tissue sections are washed and processed for the next round of analysis. The above steps of glycerol gelatin mounting, image acquisition, gentle coverslip removal in a 37°C phosphate buffer water bath, and subsequent processing are repeated until the predetermined round is completed.

[0066] refer to Figure 7 and Figure 8 The results show that, using the processing method of this application, both large tissue sections and tissue microarrays can complete high-round continuous processing and image acquisition. In images from different representative rounds, the main tissue region still maintains an identifiable morphology, and the tissue contour and main spatial structure can be used for image registration, region localization, and subsequent quantitative analysis. For tissue microarray samples, the overall arrangement of the tissue microarray dots is well maintained, and representative tissue points can still be repeatedly identified and imaged. These results indicate that the processing method of this application is suitable for high-round continuous in-situ detection processes of large tissue sections and tissue microarray samples, and can improve the ability to maintain tissue morphology and the stability of multi-round image acquisition, showing significant advantages over conventional processing methods.

[0067] The images from different rounds shown in this embodiment are only used to illustrate the effect of this method on tissue morphology preservation and image acquisition in high-round continuous processing, and do not limit the specific staining markers, antibody combinations, detection systems or detection rounds to which this invention is applicable.

[0068] In the description of this specification, the use of terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," and "some examples" indicates that the specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0069] Although embodiments of this application have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for processing tissue sections based on anti-contact pads, characterized in that, The anti-contact pad has a window that extends through the anti-contact pad along its thickness direction. The tissue sectioning method based on the anti-contact pad includes the following steps: Prepare glass slides and tissue sections, with the tissue sections attached to the surface of the glass slides, and perform pre-treatment on the tissue sections for detection; Prepare the anti-contact pad, place the anti-contact pad on the glass slide, and ensure that the tissue section or the area to be tested is completely within the window; Add a mounting medium or imaging medium into the window; Prepare a cover glass and cover it over the anti-contact pad, so that the slide, the anti-contact pad and the cover glass cooperate to form a spacer cavity to obtain the sealing assembly; Microscopic images are acquired from the mounting assembly; After image acquisition is completed, the sealing assembly is placed in a liquid environment; After the physical properties of the mounting medium or the imaging medium in the spacer cavity have changed, the coverslip is removed, and the tissue section is then processed.

2. The tissue section processing method as described in claim 1, characterized in that, The steps of placing the mounting assembly in a liquid environment after image acquisition, and removing the coverslip after the mounting medium or imaging medium in the spacer cavity has softened, swollen, or loosened, and then performing subsequent processing on the tissue section include: The cover glass assembly is immersed in a buffer solution and placed in a water bath at a set temperature, causing the cover glass or imaging medium in the spacer cavity to soften, swell, or loosen. Then, force is applied to the cover glass in the environment of the buffer solution to separate the cover glass from the anti-contact pad.

3. The tissue section processing method as described in claim 1, characterized in that, In the step of adding the sealing material or imaging medium into the window, the amount of the sealing material or imaging medium added is determined according to the thickness of the anti-contact pad and the area of ​​the window, so that the sealing material or imaging medium substantially fills the spacer cavity.

4. The tissue section processing method as described in claim 2, characterized in that, The mounting medium comprises glycerol gelatin, the buffer solution comprises phosphate buffer, and the water bath is set to a temperature range of 36.5℃-37.5℃.

5. The tissue section processing method as described in claim 1, characterized in that, Before the steps of preparing the anti-contact pad, placing the anti-contact pad on the glass slide, and ensuring that the tissue section or area to be tested is completely within the window, the following steps are also included: Select the appropriate size of the anti-contact pad. The overall size and window size of the anti-contact pad are determined based on at least two of the following: the size of the tissue section or the size of the area to be tested, the size of the coverslip, the size of the slide, the thickness of the tissue section, the viscosity of the mounting medium, and the requirements of the focal plane of the microscopy scan.

6. The tissue section processing method as described in claim 5, characterized in that, The distance between the edge of the tissue slice or the area to be detected and the inner edge of the window is in the range of 0.5mm-5mm. The distance d between the outer side of the anti-contact pad and the inner edge of the window is in the range of 0.3mm≤d≤8mm. The thickness h of the anti-contact pad is in the range of 20μm≤h≤500μm.

7. The tissue section processing method as described in claim 6, characterized in that, The distance d between the outer side of the anti-contact pad and the inner edge of the window is in the range of 0.5mm≤d≤5mm, and the thickness h of the anti-contact pad is in the range of 50μm≤h≤150μm.

8. The tissue section processing method as described in claim 1, characterized in that, The step of preparing the anti-contact pad, placing the anti-contact pad on the glass slide, and ensuring that the tissue section or area to be tested is completely within the window includes: An adhesive layer is provided on the surface of the anti-contact pad for bonding with the glass slide, and the anti-contact pad is bonded to the glass slide through the adhesive layer to fix the anti-contact pad.

9. The tissue section processing method as described in claim 1, characterized in that, After the physical properties of the mounting medium or imaging medium within the spacer cavity change, the coverslip is removed, and subsequent processing steps are performed on the tissue section, the method further includes the following steps: After the tissue section is processed for the next round of detection, the operations of adding the mounting medium or the imaging medium, covering the coverslip, image acquisition, and removing the coverslip are repeated. The above steps are repeated multiple times until the predetermined round of detection is completed.

10. The tissue section processing method as described in claim 1, characterized in that, The pretreatment of the tissue sections for detection includes: When the tissue section is a formalin-fixed paraffin-embedded tissue section, the pre-detection treatment includes dewaxing, hydration, and antigen retrieval; when the tissue section is a frozen tissue section, the pre-detection treatment includes fixation and washing.

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