Freezing section method for space transcriptomics analysis of mesentery and regenerative intestinal tract of stichopus japonicus

Abstract

The invention provides a frozen section method for space transcriptomics analysis of mesentery and regenerative intestinal tract of stichopus japonicus, and belongs to the technical field of biotechnology. The method comprises the steps of sample treatment and embedding, pre-cooling by a freezing slicer, freezing and slicing, and flaking. According to the frozen section method for space transcriptome analysis of the mesentery and the regenerative intestinal tract of the stichopus japonicus, provided by the invention, a fresh material is directly embedded, and a plurality of thin tissue blocks with the same size are integrated into a standard tissue block of 1cm, so that the high coverage rate of a tissue cross section in a space transcriptome chip capture area is ensured; rNA degradation is effectively prevented, and finally, the cDNA library is successfully constructed and is used for subsequent analysis of a space transcriptome. The technical scheme has the characteristics of simplicity and convenience in operation and stable effect, and is particularly suitable for space transcriptomics frozen section preparation of thin film type tissues.

Description

Technical Field

[0001] The present invention relates to a frozen section method, in particular to a frozen section method for spatial transcriptomic analysis of sea cucumber mesentery and regenerated intestine, belonging to the field of biotechnology. Background Art

[0002] Sea cucumber ( Apostichopus japonicus The sea cucumber ( Echinodermata ) is a representative species of the phylum Echinodermata, occupying a unique position in the evolutionary transition from invertebrates to vertebrates and widely considered an ideal model for studying phylogenetic evolution. Sea cucumbers possess a remarkable regenerative capacity. When faced with an unfavorable environment or predators, they expel their intestines, respiratory tract, gonads, and other internal organs through regurgitation. When conditions are favorable, they can rapidly regenerate a fully functional set of internal organs. The process of intestinal regeneration in sea cucumbers closely resembles embryonic development, yet is completely independent in time and space. Compared to other regeneration models, regurgitation is highly reproducible and manageable, and can be induced by artificial injection of potassium chloride, avoiding the risk of surgical trauma that could interfere with research results. These characteristics make sea cucumbers a unique model for studying visceral organ regeneration. The mesentery is widely considered the center of intestinal regeneration in sea cucumbers, and the morphological and histological changes during regeneration are subdivided into six stages: wound repair, primordium formation, lumen rudiment formation, lumen penetration, functional differentiation, and growth. The formation and development of these regenerative stages are driven by a series of orderly cellular events (cell migration, cell dedifferentiation, cell proliferation, cell death, cell redifferentiation, etc.). Research has shown that understanding the molecular mechanisms of these cellular phenomena can provide important insights for a deeper understanding of the regulatory mechanisms of metazoan regeneration.

[0003] Currently, research on the molecular mechanisms of sea cucumber intestinal regeneration has largely focused on single-gene functional analysis and global transcriptome analysis. However, traditional transcriptomics techniques have significant limitations, preventing them from obtaining spatial information about genes at the single-cell level. In contrast, spatial transcriptomics, by integrating imaging and sequencing technologies, can provide more comprehensive spatial information about gene expression, which is crucial for uncovering complex regeneration mechanisms. In tissue regeneration research, especially in complex biological processes like sea cucumber intestinal regeneration, the spatial distribution of cells and their responses to environmental changes are crucial. A deeper understanding of intercellular communication mechanisms and the differentiation of distinct cell populations are essential for constructing gene expression maps for specific tissues or organs, delineating tissue architecture, and clarifying their component elements, thus helping to better understand the dynamics of tissue changes. Spatial transcriptomics techniques enable simultaneous investigation of tissue localization and the transcriptome of individual cells, providing powerful support for studying spatial heterogeneity, intercellular communication, and interactions. Combining spatial transcriptomics with single-cell sequencing allows for the precise mapping of gene expression across tissues and regenerating tissue sections. This approach not only enables visualization of gene expression locations but also lays a solid foundation for subsequent in-depth analysis.

[0004] Cryosection preparation is a crucial and fundamental step in spatial transcriptomic analysis of the sea cucumber mesentery and regenerating intestine. Commercial detection platforms (such as Stereo-seq) require that tissue coverage of the capture area should reach 50%–100%. Existing cryosection methods primarily target three-dimensional bulk tissues, which generally meet these coverage requirements. However, the sea cucumber mesentery and early regenerating intestine are thin-film tissues (only micrometers thick), and their cross-section coverage on microarrays is difficult to meet the standards for spatial transcriptomic analysis. Furthermore, post-sampling challenges of the sea cucumber mesentery and regenerating intestine include clumping and difficulty in morphological fixation. Therefore, the establishment of a comprehensive frozen section preparation technology system is urgently needed. The establishment of such a system will not only advance the study of the molecular mechanisms of sea cucumber intestinal regeneration but also provide an important reference for the preparation of spatial transcriptomic samples from other thin-film-like tissues. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical problems and provide a frozen section method for spatial transcriptomic analysis of the mesentery and regenerated intestine of sea cucumbers. The tissue sections prepared by the present invention have the advantages of tissue coverage meeting spatial transcriptomic analysis and clear cell structure.

[0006] To achieve the above object, the technical solution adopted by the present invention is: One of the objectives of the present invention is to first provide a frozen section method for spatial transcriptomic analysis of the mesentery and regenerated intestine of sea cucumbers, comprising the following steps: (1) Sample processing and embedding: ① Take the mesentery and regenerated intestine of the sea cucumber with a length and width of 1 cm each, wash them with PBS, and then use flat-head forceps to hold one side of the mesentery and regenerated intestine and immerse them in ice-cold OCT embedding medium; ② Confirm the direction of tissue sectioning: Drag the sea cucumber mesentery and regenerated intestine into the embedding box with OCT embedding medium again, so that the mesentery and regenerated intestine are flattened in the embedding box, and then drop OCT embedding medium that can just cover the tissue, and confirm that there are no bubbles near the tissue; ③Immediately place the embedding cassette in liquid nitrogen or a -80°C freezer until the OCT embedding medium is completely frozen; ④ Repeat the embedding of multiple sea cucumber mesentery and regenerated intestinal samples, take out the embedded frozen blocks, add OCT embedding medium, stack multiple frozen blocks in parallel and stick them together, and then quickly freeze them in liquid nitrogen to obtain tissue embedding blocks; (2) Precooling of the cryostat: The temperature of the cryostat freezer is set to -20°C, and the temperature of the quick-freezing table is set to -20°C; (3) Block trimming: The tissue embedding block obtained in step (1) is appropriately trimmed to remove excess OCT embedding agent and ensure that the sections of the embedding block remain parallel; then, OCT embedding solution is applied to the sample holder as a base, and the trimmed embedding block is accurately positioned and then transferred to a -20°C quick-freezing table for 10 minutes to fix the embedding block on the sample holder; (4) Sectioning: After adjusting the angle of the sample freezing block, trim the freezing block, and then perform continuous sectioning to collect the sections that meet the requirements.

[0007] In the above technical solution, in step (1), the mesentery and regenerated intestine are tissues of the sea cucumber intestine regeneration stage 0-28 days.

[0008] In the above technical solution, in step (1), the OCT embedding agent is SAKURA Tissue-Tek® OCT Compound, Order Number 4583.

[0009] In the above technical solution, in step (1), the mesentery and regenerated intestinal tissue are flattened into the embedding box so that the mesentery and regenerated intestinal tissue are parallel to the bottom surface of the embedding box.

[0010] In the above technical solution, in step (1), after the embedding block is completely frozen, if the mesentery and regenerated intestine are exposed and not completely embedded through the OCT embedding agent, continue to add OCT embedding agent, but ensure that the thickness of each piece of mesentery and regenerated intestine frozen embedding sample does not exceed 0.2 cm.

[0011] In the above technical solution, in step (1), all prepared mesentery and regenerated intestine frozen embedding blocks have the same length and width.

[0012] In the above technical solution, in step (3), the embedded mesentery and regenerated intestinal frozen block needs to be trimmed before being placed on the sample holder to remove excess OCT embedding agent.

[0013] In the above technical solution, in step (4), when performing continuous sectioning, the section thickness is first set to 50 μm, and the excess tissue is quickly removed. When the section surface is close to the sample to be collected, the section thickness is set to 10 μm, and the sample morphology in the section is observed. If it meets the requirements, the sample section is collected.

[0014] In the above technical solution, in step (4), after collecting the slices that meet the requirements, the slices are flattened and attached to the detection area of ​​the Stereo-seq slide. After being fixed with paraformaldehyde and air-dried, the slices are quickly transferred to a -80°C refrigerator for storage.

[0015] A second object of the present invention is to provide a frozen section prepared by the above-mentioned frozen section method for use in spatial transcriptomic analysis of sea cucumber mesentery and regenerated intestinal tissue.

[0016] The third object of the present invention is to provide an application of the frozen sections for spatial transcriptomic analysis of the sea cucumber mesentery and regenerated intestine in spatial transcriptomic analysis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides an improved frozen section method suitable for spatial transcriptomic analysis of the mesentery and regenerating intestine of sea cucumbers. This method uses a direct embedding technique of fresh materials. By integrating multiple thin tissue blocks of the same size into a standard tissue block of 1 cm³, it ensures high coverage of the tissue cross section in the capture area of ​​the spatial transcriptome chip and effectively prevents RNA degradation, ultimately successfully constructing a cDNA library. This technical solution is characterized by simple operation and stable results, and is particularly suitable for the preparation of frozen sections for spatial transcriptomic analysis of thin-film tissues.

[0018] (2) The present invention provides a frozen section prepared by the above method for spatial transcriptomic analysis of the mesentery and regenerated intestine of sea cucumbers, which provides a technical basis for spatial transcriptomic analysis of the mesentery and regenerated intestine of sea cucumbers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description: Figure 1 This is HE staining imaging of frozen sections of the sea cucumber mesentery and regenerated intestine obtained in Example 1 of the present invention; Figure 2The invention is the present invention. The frozen section of the sea cucumber mesentery and regenerated intestine is obtained by fluorescence imaging. Figure 3 The results of the construction and detection of the cDNA library of frozen sections of the sea cucumber mesentery and regenerated intestine obtained in embodiment 1 of the present invention are as follows: the cDNA fragment range is 593-1940 bp, the main peak is 1102 bp, the fragment range of the interrupted product is within the normal range, and there is no abnormality in the library construction. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the following examples further describe the operation of the present invention in detail. The following examples are only used to illustrate the invention but are not intended to limit the scope of the invention.

[0021] The present invention provides a frozen section method for spatial transcriptomic analysis of the mesentery and regenerated intestine of sea cucumbers, comprising the following steps: (1) Sample processing and embedding: ① Take the mesentery and regenerated intestine of the sea cucumber with a length and width of 1 cm each, wash them with PBS, and then use flat-head forceps to hold one side of the mesentery and regenerated intestine and immerse them in ice-cold OCT embedding medium; ② Confirm the direction of tissue sectioning: Drag the sea cucumber mesentery and regenerated intestine into the embedding box with OCT embedding medium again, so that the mesentery and regenerated intestine are flattened in the embedding box, and then drop OCT embedding medium that can just cover the tissue, and confirm that there are no bubbles near the tissue; ③Immediately place the embedding cassette in liquid nitrogen or a -80°C freezer until the OCT embedding medium is completely frozen; ④ Repeat the above embedding of multiple sea cucumber mesentery and regenerated intestinal samples, take out the embedded frozen blocks, add OCT embedding medium, stick the multiple frozen blocks together and quickly freeze them in liquid nitrogen; (2) Precooling of the cryostat: The temperature of the cryostat freezer is set to -20°C, and the temperature of the quick-freezing table is set to -20°C; (3) Preparation of sample frozen blocks: squeeze OCT embedding solution onto the sample holder as a base, place the embedded mesentery and the regenerated intestinal frozen block at its end on the sample holder, and then transfer it to a quick-freeze table that has been pre-cooled to -20°C and let it stand. Fix the sample on the sample holder; (4) Sectioning: After adjusting the angle of the sample freezing block, trim the freezing block, and then perform continuous sectioning to collect the sections that meet the requirements.

[0022] The present invention also provides a frozen section prepared by the frozen section method for spatial transcriptomic analysis of sea cucumber mesentery and regenerated intestinal tissue.

[0023] The present invention also provides a use of the frozen section for spatial transcriptomic analysis of the sea cucumber mesentery and regenerated intestine in spatial transcriptomic analysis.

[0024] The technical solution of the present invention is described below in conjunction with specific embodiments: Example 1

[0025] This embodiment provides a frozen section method for spatial transcriptomic analysis of the mesentery and regenerating intestine of sea cucumbers, comprising the following steps: (1) Sample processing and embedding: The sea cucumber mesentery and regenerated intestine used in this example were tissues from the sea cucumber intestinal regeneration stage 0-28 days; the OCT embedding medium was SAKURA Tissue-Tek® OCT Compound, Order Number 4583.

[0026] ① Take 1 cm long and 1 cm wide sea cucumber mesentery and regenerated intestine, wash them with PBS, and then use flat-tip forceps to hold one side of the mesentery and immerse it in ice-cold OCT embedding medium to remove excess PBS; ② Then, drag the mesentery and regenerated intestine into the embedding box with pre-cooled OCT embedding medium again, so that the mesentery and regenerated intestine are flattened in the embedding box. Adjust the direction of the tissue section, and then drop OCT embedding medium just enough to cover the tissue. Make sure there are no bubbles near the tissue. ③ Note the sample name on the embedding box, and then immediately place the embedding box in liquid nitrogen or a -80℃ freezer until the OCT embedding medium is completely frozen; ④ Repeat the above embedding of multiple sea cucumber mesentery and regenerated intestinal samples; take out the embedded frozen blocks, add OCT embedding agent, stick multiple frozen blocks together and quickly freeze them in liquid nitrogen.

[0027] (2) Precooling of the cryostat: Start the freezing microtome (LEICA CM1950), set the freezing chamber temperature to -20°C, set the quick freezing table temperature to -20°C, and start subsequent operations after all parts of the instrument reach the set temperature.

[0028] (3) Repairing blocks: The tissue embedding block was trimmed appropriately to remove excess OCT embedding agent and ensure that the sections of the embedding block remained parallel. OCT embedding agent was applied to the specimen holder as a base. After the trimmed embedding block was precisely positioned, it was transferred to a -20°C quick freezer and frozen for 10 minutes to fix the embedding block on the specimen holder.

[0029] (4) Slicing: Secure the specimen to the specimen holder, adjust the position of the slide frame and blade, and then proceed with sectioning. First, set the section thickness to 50 μm and quickly remove excess tissue from the embedded block. When the section approaches the sample to be collected, set the section thickness to 10 μm. While slicing, use an adhesive slide to absorb the section. Preliminarily observe the sample outline to determine whether to start sample collection. After collecting the sections that meet the requirements, flatten them and attach them to the detection area of ​​the Stereo-seq slide. After fixation with 4% paraformaldehyde and air drying, immediately transfer to a -80°C freezer for storage.

[0030] HE staining observation was performed on the frozen sections collected in accordance with the requirements of the embodiment of the present invention: HE staining imaging of the sections was as follows: Figure 1 As shown by Figure 1 It can be seen that the mesentery and regenerated intestine at different regeneration stages are in the same cross-section, and HE staining observes that the tissue structure is complete and clear.

[0031] Permeabilization gradient fluorescence imaging analysis was performed on the frozen sections collected in accordance with the requirements of the present invention: the results showed that treatment with 0.3% permeabilization enzyme for 12 minutes can maximize RNA release while maintaining tissue morphological integrity (RIN = 7.9). Figure 2 As shown, PolyA probe fluorescence imaging showed that the entire slice presented uniform bright light, confirming that RNA was completely released.

[0032] The cDNA library was constructed for the frozen sections that met the requirements collected in the embodiment of the present invention: the cDNA library was constructed for the sea cucumber mesentery and regenerated intestinal tissue sections using the Stereo-seq platform. Figure 3 As shown: the cDNA fragment range is 593-1940 bp, the main peak is 1102 bp, the fragment range of the sheared product is within the normal range, and there is no abnormality in the library construction.

[0033] The above examples are only for illustrating the technical concept and technical features of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent transformation or modification made based on the essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A frozen section method for spatial transcriptomic analysis of the mesentery and regenerated intestine of sea cucumbers, characterized in that: The following steps are involved: (1) Sample processing and embedding: ① Take the mesentery and regenerated intestine of the sea cucumber with a length and width of 1 cm each, wash them with PBS, and use flat-head forceps to hold one side of the mesentery and regenerated intestine and immerse them in ice-cold OCT embedding medium; ② Confirm the direction of tissue sectioning: Drag the sea cucumber mesentery and regenerated intestine into the embedding box with OCT embedding medium again, so that the mesentery and regenerated intestine are flattened in the embedding box, and then drop OCT embedding medium that can just cover the tissue, and confirm that there are no bubbles near the tissue; ③Immediately place the embedding cassette in liquid nitrogen or a -80°C freezer until the OCT embedding medium is completely frozen; ④ Repeat the above embedding of multiple sea cucumber mesentery and regenerated intestinal samples, take out the embedded frozen blocks, add OCT embedding medium, stick the multiple frozen blocks together and quickly freeze them in liquid nitrogen to obtain tissue embedding blocks; (2) Precooling of the cryostat: The temperature of the cryostat freezer is set to -20°C, and the temperature of the quick-freezing table is set to -20°C; (3) Block trimming: The tissue embedding block obtained in step (1) is appropriately trimmed to remove excess OCT embedding agent and ensure that the sections of the embedding block remain parallel; OCT embedding agent is applied to the sample holder as a base, and the trimmed embedding block is accurately positioned and transferred to a -20°C quick-freezing table for 10 minutes to fix the embedding block on the sample holder; (4) Sectioning: After adjusting the angle of the sample freezing block, trim the freezing block, and then perform continuous sectioning to collect the sections that meet the requirements.

2. The frozen section method according to claim 1, wherein In step (1), the mesentery and regenerated intestine are tissues of sea cucumber intestine regeneration stage 0-28 days; the OCT embedding agent is SAKURA Tissue-Tek® OCT Compound, Order Number 4583.

3. The frozen section method according to claim 1, wherein In step (1), the mesentery and regenerated intestinal tissue are flattened into an embedding box so that the mesentery and regenerated intestinal tissue are parallel to the bottom surface of the embedding box.

4. The frozen section method according to claim 1, wherein In step (1), after the embedding block is completely frozen, if the mesentery and regenerated intestine are exposed through the OCT embedding agent and are not completely embedded, continue to add OCT embedding agent, but ensure that the thickness of each frozen tissue embedding sample does not exceed 0.2 cm.

5. The frozen section method according to claim 1, wherein In step (1), all prepared mesentery and regenerated intestine frozen embedding blocks have the same length and width.

6. The frozen section method according to claim 1, wherein: In step (3), the embedded mesentery and regenerated intestinal frozen block needs to be trimmed before being placed on the sample tray to remove excess OCT embedding medium.

7. The frozen section method according to claim 1, wherein In step (4), when performing continuous sectioning, first set the section thickness to 50 μm, quickly remove excess tissue, and when the section surface is close to the sample to be collected, set the section thickness to 10 μm and slice, observe the sample morphology in the section, and collect the sample section after it meets the requirements.

8. The frozen section method according to claim 1, wherein: In step (4), after collecting the slices that meet the requirements, the slices are flattened and attached to the rough surface of an adhesive slide placed at room temperature. After the OCT embedding agent quickly melts so that the slices adhere to the slide, the slices are quickly transferred to a -80°C refrigerator for storage.

9. A frozen section prepared by the frozen section method according to any one of claims 1 to 8 for use in spatial transcriptomic analysis of sea cucumber mesentery and regenerated intestinal tissue.

10. Use of the frozen section for spatial transcriptomic analysis of sea cucumber mesentery and regenerated intestine according to claim 8 in spatial transcriptomic analysis.

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