A paraffin tissue section anti-falling piece method suitable for CosMxSMI space transcriptome analysis

Abstract

The application discloses a paraffin tissue section anti-falling piece method suitable for CosMx SMI spatial transcriptome analysis. The application optimizes key steps such as section spreading, section sticking, section segment baking, water bath heat treatment, overnight dewaxing, drying after antigen repair and low-concentration protease permeation, improves the adhesion stability of FFPE tissue sections in the whole process of CosMx SMI, and takes into account the compatibility of tissue morphology maintenance, RNA signal detection and subsequent multiple rounds of fluorescence imaging. The application is suitable for FFPE tissue sections, especially for tumor tissues, inflammatory tissues, fibrotic tissues, clinical archival tissues and paraffin samples with high risk of falling.

Description

Technical Field

[0001] This invention relates to the field of slide removal technology, specifically to a method for preventing slide removal from paraffin tissue sections suitable for CosMx SMI spatial transcriptome analysis. Background Technology

[0002] The CosMx Spatial Molecular Imager (SMI) is a spatial molecular imaging analysis platform based on in situ hybridization, cyclic reporter probe hybridization, and high-resolution fluorescence imaging readout. It can detect and quantify multiple RNA and protein targets in situ in formalin-fixed paraffin-embedded (FFPE) tissue sections or fresh frozen sections. The sample preparation stage typically includes section fixation and permeabilization, RNA probe hybridization, washing, Oligo-labeled morphological antibody hybridization, flow cell assembly, field of view (FOV) selection, and multiple rounds of reporter probe hybridization and fluorescence imaging at the instrument.

[0003] Compared to conventional hematoxylin-eosin staining, immunohistochemistry, or single-round immunofluorescence, CosMx SMI places higher demands on the tissue adhesion stability of FFPE sections. This is because: firstly, the sections need to undergo dewaxing, rehydration / dehydration, high-temperature antigen retrieval, protease permeation, and multiple washing processes; secondly, they need to be combined with flow cells and undergo multiple rounds of liquid exchange, reporter probe hybridization, and imaging within the instrument; and thirdly, spatial transcriptomics experiments typically require maintaining the spatial correspondence between tissue structure, cell boundaries, RNA signal sites, and morphological markers. If tissue sections warp, shrink, float, or detach, it will directly lead to loss of the region of interest (ROI), inaccurate cell segmentation, decreased transcript counts, or even failure of the entire section experiment.

[0004] Current FFPE slide sample preparation typically employs conventional pathological mounting and baking procedures, such as spreading the slide at 42°C, attaching it to a glass slide, and then baking it at 50-60°C once or performing conventional dewaxing. While this type of procedure can meet the needs of general histological staining, it has the following drawbacks in long-process spatial transcriptomics experiments like CosMx SMI, which involve multiple rounds of liquid exchange and high-temperature / enzymatic digestion: conventional single-use baking or dewaxing is insufficient to withstand the high-temperature antigen retrieval, protease permeation, prolonged liquid exchange, and flow cell assembly operations in the CosMx SMI procedure, resulting in the risk of edge curling, local detachment, or overall detachment of the slides; simply extending the baking time may enhance tissue adhesion, but it can easily lead to excessive tissue drying, decreased RNA preservation quality, impaired morphological antibody signals, or reduced efficiency of subsequent probe entry into the tissue; relying solely on improving slide adhesion cannot adequately address the stress changes between dewaxing, antigen retrieval, ethanol dehydration, and protease digestion, and in particular, cannot stably control the adhesion state of the slides at each processing stage; existing CosMx SMI standard procedures emphasize probe hybridization, imaging, and data reading, lacking targeted optimization of pretreatment parameters for preventing FFPE slide detachment, which is not conducive to obtaining stable results from different batches of FFPE samples.

[0005] In summary, FFPE slides obtained using existing techniques may exhibit issues such as tissue edge lifting, tissue fragment detachment, localized area loss, and uneven adhesion between the slide and the slide. The risk of slide detachment is particularly high for FFPE samples with large tissue areas, significant fibrosis, numerous necrotic areas, high fat content, or those preserved for extended periods.

[0006] Therefore, there is an urgent need to establish a method for preventing detachment of FFPE sections that is compatible with the CosMx SMI standard sample processing workflow, is easy to operate, does not require changes to the instrument's readout logic, does not require the introduction of complex tissue expansion or removal steps, and can significantly improve the adhesion stability of FFPE sections. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preventing detachment of paraffin-embedded tissue sections suitable for CosMx SMI spatial transcriptomics analysis. This invention improves the adhesion stability of FFPE tissue sections throughout the CosMx SMI process by optimizing key steps such as slide spreading, mounting, segmented baking, water bath heat treatment, overnight dewaxing, antigen retrieval followed by drying, and low-concentration protease permeation. It also ensures compatibility with tissue morphology preservation, RNA signal detection, and subsequent multiple rounds of fluorescence imaging. This invention is applicable to FFPE tissue sections, especially tumor tissues, inflammatory tissues, fibrotic tissues, clinically archived tissues, and paraffin samples with a high risk of detachment that require CosMx SMI spatial transcriptomics or spatial multi-omics analysis.

[0008] The objective of this invention is achieved as follows:

[0009] A method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis includes the following steps:

[0010] S1. Prepare paraffin sections from FFPE tissue, spread them in a water bath at 40-45℃, and then attach them to a glass slide to obtain a mounted glass slide.

[0011] S2. The slides obtained in S1 are subjected to segmented heat treatment: first, the slides are baked at 65°C for 2 hours, then treated in a water bath at 62°C for 1 hour, and then baked at 65°C for 2 hours to obtain heat-treated slides.

[0012] S3. Place the heat-treated slices obtained in S2 at 60°C for at least 16 hours to dewax them, and obtain the dewaxed slices.

[0013] S4. The dewaxed sections obtained in S3 are washed sequentially with xylene as the first cleaning solution, xylene as the second cleaning solution, 100% ethanol as the third cleaning solution, and 100% ethanol as the fourth cleaning solution. The sections are then dried and placed at room temperature to equilibrate, resulting in sections after the first cleaning.

[0014] S5. Place the slices obtained after one washing in S4 into preheated 1×CosMx retrieval buffer antigen retrieval solution and treat them in a water bath at 100℃ for 15 min to obtain antigen-retrieval slices.

[0015] S6. After antigen retrieval, the slide obtained in S5 is transferred to fresh DEPC water and washed by moving it up and down for 15 seconds. Then it is placed in 100% ethanol for 3 minutes, and then transferred to a clean, low-dust, low-fiber absorbent wiping material and dried at room temperature for 30-60 minutes to obtain the slide after secondary washing.

[0016] S7. Using a hydrophobic histological pen, draw a closed line around the tissue area of ​​the section obtained after secondary cleaning in S6 to form a closed hydrophobic fence; add room temperature digestion buffer to the area enclosed by the hydrophobic fence so that the digestion buffer completely covers the tissue area to obtain a first-processed section.

[0017] S8. Place the first-processed slices obtained in S7 in a humidified box preheated to 40°C and incubate in a 40°C oven for 30 minutes to obtain the second-processed slices.

[0018] S9. Transfer the secondary processed slices obtained in S8 to DEPC water and wash them twice, moving the slices up and down 3-5 times each time. After washing, the slices are temporarily stored in DEPC water and then enter the CosMx SMI standard process.

[0019] In S1, the thickness of the paraffin slice is 4-6 μm.

[0020] In step S4, the cleaning time with xylene in the first cleaning solution is 5 minutes; the cleaning time with xylene in the second cleaning solution is 5 minutes; the cleaning time with 100% ethanol in the third cleaning solution is 2 minutes; and the cleaning time with 100% ethanol in the fourth cleaning solution is 2 minutes.

[0021] In step S4, the product is dried at 60°C for 5 minutes.

[0022] In step S5, the 1×CosMx retrieval buffer antigen retrieval solution is preheated at 100°C for 15 minutes.

[0023] In step S7, the Digestion buffer is prepared by diluting a 20 mg / mL Proteinase K stock solution to a working concentration of 3 μg / mL and dissolving it in 1×PBS.

[0024] The beneficial effects of this invention are: 1. This invention can significantly improve the tissue integrity of FFPE tissue sections during pretreatment, and reduce the risk of detachment, edge curling and local tissue defects; it is especially suitable for spatial transcriptome analysis of large tissue areas, loose structures or precious clinical FFPE samples.

[0025] 2. This invention improves the integrity of tissue sections, thereby increasing the number of effective imaging fields and analyzable tissue area after samples enter the CosMx SMI standard process, thus enhancing the success rate of spatial transcriptomics detection and data acquisition efficiency. Specifically, through techniques such as segmented baking, water bath heat treatment, overnight dewaxing, ethanol treatment after antigen retrieval and room temperature drying, gentle permeation with low-concentration Proteinase K, and gentle washing with DEPC water, the risk of tissue sections detaching during pretreatment and instrumentation is reduced. Attached Figure Description

[0026] Figure 1 This is a comparison of the tissue retention effects of the optimized anti-detachment method of this invention and the traditional method after processing FFPE tissue sections;

[0027] Figure 2 This is a comparison chart showing the effective FOV of samples processed by the optimized method of this invention and the traditional method before entering the SMI standard process. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] A method for preventing detachment of paraffin tissue sections for CosMx SMI spatial transcriptomics analysis is proposed. This method is applicable to FFPE tissue sections, especially to tumor tissues, inflammatory tissues, fibrotic tissues, clinical archived tissues, and paraffin samples with a high risk of detachment that require CosMx SMI spatial transcriptomics or spatial multi-omics detection.

[0030] Includes the following steps:

[0031] S1. Prepare paraffin sections with a thickness of 5 μm from FFPE tissue, spread them in a 42℃ water bath, and then attach them to a glass slide to obtain a mounted glass slide. Spreading and mounting the paraffin sections in a 42℃ water bath ensures that the paraffin sections are fully expanded, reducing wrinkles and local tension; the temperature of spreading and mounting are consistent to reduce tissue shrinkage and edge lifting caused by sudden temperature changes.

[0032] S2. The slides obtained in S1 are subjected to segmented heat treatment: First, the slides are baked at 65℃ for 2 hours to promote initial stable bonding between the slides and the slide and to remove local water. Then, they are treated in a 62℃ water bath for 1 hour to further smooth and adhere the tissue to the slide through a mild heat and humidity environment, buffering the excessive drying caused by simple dry baking. Subsequently, the slides are baked at 65℃ for another 2 hours to further strengthen tissue adhesion and provide a stable foundation for subsequent long-term dewaxing and high-temperature repair. Heat-treated slides are obtained.

[0033] S3. Place the heat-treated sections obtained in S2 at 60°C for at least 16 hours to dewax them, and obtain dewaxed sections; soften and remove some of the paraffin before entering xylene to reduce tissue buoyancy during subsequent organic solvent treatment;

[0034] S4. The dewaxed sections obtained in S3 were sequentially washed with xylene (first washing solution), xylene (second washing solution), 100% ethanol (third washing solution), and 100% ethanol (fourth washing solution) to complete dewaxing and dehydration, ensuring the subsequent entry of repair solution, probes, and antibodies into the tissue. The sections were then dried at 60℃ for 5 minutes and allowed to equilibrate at room temperature to remove residual ethanol and surface liquid, making the sections more stable and preventing localized peeling caused by direct exposure to high temperatures for repair. The sections were obtained after one washing cycle; the xylene washing time was 5 minutes for the first cycle; the xylene washing time was 5 minutes for the second cycle; the 100% ethanol washing time was 2 minutes for the third cycle; and the 100% ethanol washing time was 2 minutes for the fourth cycle.

[0035] S5. Place the washed sections obtained in S4 into preheated 1×CosMx retrieval buffer antigen retrieval solution and incubate at 100℃ for 15 min to ensure tissue permeation and target exposure, while preheating the retrieval solution to reduce temperature fluctuations. Obtain antigen-retrieval sections; 1×CosMx retrieval buffer antigen retrieval solution preheated at 100℃ for 15 min;

[0036] S6. After antigen retrieval, the slides obtained in S5 are transferred to fresh DEPC water and washed by moving them up and down for 15 seconds to remove the retrieval solution and reduce residual salt content, while controlling mechanical disturbance. They are then placed in 100% ethanol for 3 minutes, and then transferred to a clean, low-dust, low-fiber absorbent wiping material. They are then dried at room temperature for 30-60 minutes to form a stable dry state before protease permeation, reducing the risk of slide bleed or edge peeling caused by local water films. The slides are then obtained after the second washing.

[0037] S7. Using a hydrophobic histochemical pen, draw a closed line around the tissue area of ​​the section obtained after secondary washing in S6 to form a closed hydrophobic fence; add room temperature digestion buffer to the area enclosed by the hydrophobic fence so that the digestion buffer completely covers the tissue area to obtain a first-processed section; the digestion buffer is made by diluting 20 mg / mL Proteinase K stock solution to a working concentration of 3 μg / mL and dissolving it in 1×PBS.

[0038] S8. Place the first-processed sections obtained in S7 in a humidified chamber preheated to 40°C and incubate in a 40°C oven for 30 min to obtain the second-processed sections; digest at a lower working concentration and controlled temperature to balance tissue permeability, RNA detection and tissue adhesion.

[0039] S9. Transfer the secondary processed slices obtained in S8 to DEPC water and wash twice, moving the slices up and down 3-5 times each time to remove protease and buffer residues. After washing, the slices are temporarily stored in DEPC water and then proceeded to the CosMx SMI standard procedure.

[0040] Analysis of experimental cases:

[0041] Compared with FFPE tissue sections processed by conventional methods, the present invention is as follows: Figure 1As shown, after processing FFPE tissue sections using traditional methods, some samples exhibited edge lifting, local fragmentation, pore-like defects, or tissue detachment. This indicates that conventional mounting, baking, and dewaxing methods are insufficient to fully withstand the mechanical and thermal stress caused by subsequent organic solvent washing, high-temperature antigen retrieval, and protease permeation steps. This invention utilizes a combination of "42℃ water bath spreading and mounting, 65℃ first baking, 62℃ water bath heat treatment, 65℃ second baking, and 60℃ overnight dewaxing" to ensure that paraffin sections are fully expanded, flattened, and gradually enhanced in their adhesion to the glass slide before entering the xylene, ethanol, and high-temperature retrieval steps. Simultaneously, rapid washing with DEPC water after antigen retrieval, treatment with 100% ethanol, and room temperature drying stabilize the sections before protease permeation, reducing the risk of section drift or edge peeling caused by local water films, temperature fluctuations, and liquid disturbance. Therefore, this invention can improve the preservation of the integrity of the main tissue structure and reduce the risk of section detachment during sample pretreatment, making it particularly suitable for spatial transcriptome analysis of large, loosely structured, or valuable clinical FFPE samples. Figure 1 The serial numbers 1 / 6, 2 / 7, 3 / 8, and 5 / 9 represent two slices of the same sample.

[0042] like Figure 2 As shown, samples processed using traditional methods, upon entering the SMI standard workflow, suffer from varying degrees of tissue detachment, fragmentation, edge curling, or localized defects during pretreatment. This results in a relatively small number of effective FOVs that can be identified and included in imaging analysis, and some tissue areas struggle to form continuous and stable analyzable imaging regions. In contrast, samples processed using the optimized anti-detachment method of this invention retain the main tissue structure more stably on the slide surface during dewaxing, antigen retrieval, ethanol treatment, protease permeation, and subsequent washing. Upon entering the SMI standard workflow, they maintain a more complete tissue coverage, thus significantly increasing the number of effective FOVs, resulting in a more continuous FOV distribution and a larger analyzable tissue area. This beneficial effect primarily stems from the invention's systematic reduction of the risk of tissue section detachment during pretreatment and instrumentation through its technical features, including segmented baking, water bath heat treatment, overnight dewaxing, ethanol treatment and room temperature drying after antigen retrieval, gentle permeation with low-concentration Proteinase K, and gentle washing with DEPC water. Therefore, this invention can reduce invalid FOV, duplicate experiments and sample waste caused by tissue shedding or local defects, and improve the effective imaging range, data acquisition efficiency and overall success rate of CosMx SMI spatial transcriptomics experiments.

Claims

1. A method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis, characterized in that, Includes the following steps: S1. Prepare paraffin sections from FFPE tissue, spread them in a water bath at 40-45℃, and then attach them to a glass slide to obtain a mounted glass slide. S2. The slides obtained in S1 are subjected to segmented heat treatment: first, the slides are baked at 65°C for 2 hours, then treated in a water bath at 62°C for 1 hour, and then baked at 65°C for 2 hours to obtain heat-treated slides. S3. Place the heat-treated slices obtained in S2 at 60°C for at least 16 hours to dewax them, and obtain the dewaxed slices. S4. The dewaxed sections obtained in S3 are washed sequentially with xylene as the first cleaning solution, xylene as the second cleaning solution, 100% ethanol as the third cleaning solution, and 100% ethanol as the fourth cleaning solution. The sections are then dried and placed at room temperature to equilibrate, resulting in sections after the first cleaning. S5. Place the slices obtained after one washing in S4 into preheated 1×CosMx retrieval buffer antigen retrieval solution and treat them in a water bath at 100℃ for 15 min to obtain antigen-retrieval slices. S6. After antigen retrieval, the slide obtained in S5 is transferred to fresh DEPC water and washed by moving it up and down for 15 seconds. Then it is placed in 100% ethanol for 3 minutes, and then transferred to a clean, low-dust, low-fiber absorbent wiping material and dried at room temperature for 30-60 minutes to obtain the slide after secondary washing. S7. Using a hydrophobic histological pen, draw a closed line around the tissue area of ​​the section obtained after secondary cleaning in S6 to form a closed hydrophobic fence; add room temperature digestion buffer to the area enclosed by the hydrophobic fence so that the digestion buffer completely covers the tissue area to obtain a first-processed section. S8. Place the first-processed slices obtained in S7 in a humidified box preheated to 40°C and incubate in a 40°C oven for 30 minutes to obtain the second-processed slices. S9. Transfer the secondary processed slices obtained in S8 to DEPC water and wash them twice, moving the slices up and down 3-5 times each time. The washed slices are temporarily stored in DEPC water and then proceed with the CosMx SMI standard process.

2. The method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis according to claim 1, characterized in that, In S1, the thickness of the paraffin slice is 4-6 μm.

3. The method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis according to claim 1, characterized in that, In step S4, the cleaning time with xylene in the first cleaning solution is 5 minutes; the cleaning time with xylene in the second cleaning solution is 5 minutes; the cleaning time with 100% ethanol in the third cleaning solution is 2 minutes; and the cleaning time with 100% ethanol in the fourth cleaning solution is 2 minutes.

4. The method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis according to claim 1, characterized in that, In step S4, the product is dried at 60°C for 5 minutes.

5. The method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis according to claim 1, characterized in that, In step S5, the 1×CosMx retrieval buffer antigen retrieval solution is preheated at 100°C for 15 minutes.

6. The method for preventing paraffin tissue sections from detaching during CosMx SMI spatial transcriptome analysis according to claim 1, characterized in that, In step S7, the Digestion buffer is prepared by diluting a 20 mg / mL Proteinase K stock solution to a working concentration of 3 μg / mL and dissolving it in 1×PBS.

Images