Preparation method of frozen section of tree tissue invaded by diseases and pests

Abstract

The invention relates to the technical field of biological experiments and plant microscopy, in particular to a preparation method of a frozen section of tree tissue invaded by diseases and pests for spatial transcriptomics analysis. Specifically, during sample collection and sample segmentation, transverse cutting is reduced, a fan-shaped sample is formed, a method of directly embedding and slicing after fixing and vacuumizing is adopted, dehydration and dyeing are not carried out, and a tree obtained after slicing is complete in tissue structure and clear in cell, so that loss of genetic materials in the cell is effectively avoided; and the method is a freezing and slicing mode with a good effect. The method is particularly suitable for preparing the frozen section of the tree tissue at the egg chamber part of the anoplophora glabripennis on the elaeagnus angustifolia in space transcriptomics analysis.

Description

Technical Field

[0001] The present invention relates to the technical fields of biological experiments and plant microscopy, and particularly relates to a method for preparing frozen sections of tree tissues damaged by pests and diseases for spatial transcriptomics analysis. Background Art

[0002] Anoplophora glabripennis, belonging to the Cerambycidae family of Coleoptera, is a major wood-boring pest of trees that has attracted global attention and damages a variety of broad-leaved trees such as poplar, willow, elm, birch, and maple. Elaeagnus angustifolia can not only efficiently attract adult Anoplophora glabripennis to feed on the bark, petioles, and leaves of 1- to 3-year-old branches to supplement nutrition, and induce female adults to gnaw grooves and lay eggs on thick branches; but also secrete a large amount of tree gum in the grooves to wrap and kill the eggs or newly hatched larvae, resulting in Anoplophora glabripennis being unable to complete its life cycle; reasonably planting Elaeagnus angustifolia can achieve ecological self-control of Anoplophora glabripennis disasters. Therefore, it is very crucial to study the response mechanism of Elaeagnus angustifolia secreting tree gum to kill Anoplophora glabripennis eggs and screen out the key genes among them.

[0003] Spatial transcriptomics combines imaging and sequencing technologies, can reveal the spatial distribution of each cell type in tissues, the interactions between cell populations, and map the gene expression profiles of different tissue regions, and has profound application value for understanding plant tissue development and defense mechanisms. However, due to the specific cell structure of plant cells themselves and the complex diversity of plant tissues, the application of spatial transcriptomics in plant tissues lags behind. Plant cells have cell walls, large vacuoles, middle lamella and other cell structures that animal cells do not have, and the existence of these cell structures poses certain experimental challenges for the application of spatial transcriptomics to plant cells, including the preparation of plant tissue sections and the guarantee of the integrity of plant cell mRNA. Plant tissue sectioning technology is used to observe the structure of plant tissues, and the tissue sections obtained by this technology can also be used to detect the expression level of genes in cells, transcription and metabolism at the cellular level, and other cell activities. The sections used in spatial transcriptomics are mainly the frozen section method. On this basis, the woody plant Elaeagnus angustifolia is extremely hard in texture, with a complex texture and structure. After being grooved and oviposited by insects such as Anoplophora glabripennis, the structure of some parts of the tree is further damaged and squeezed, making it more difficult to ensure the integrity of its tissue sections and the stability of each part of the structure.

[0004] Pest infestation can affect the tissue structure and physiological functions of trees, and its infection process usually destroys the integrity and stability of tree tissues. Many pathogenic microorganisms, including fungi (such as Verticillium dahliae), bacteria (such as Xanthomonas spp.), viruses (such as Prunus necrotic ringspot virus), phytoplasmas (such as Apple proliferation phytoplasma), and nematodes (such as Bursaphelenchus xylophilus), can grow, develop, and reproduce on the surface or inside the host trees. These pathogen infections may cause a series of pathological changes, such as mechanical damage to tree tissues, cavity formation, vascular bundle blockage, abnormal thickening of the cambium cell wall, increased gum secretion, and the formation of gum ducts. In addition, insects that use trees as their main food source or host environment can also cause similar tissue damage and pathological changes. Taking the egg-laying behavior of Anoplophora glabripennis in grooves as an example: The female adult of Anoplophora glabripennis will first use its mouthparts to bite a groove that penetrates the phloem from the outside of the host bark, then turn around, insert the ovipositor into the groove, swing it between the phloem and xylem to make an "egg chamber", lay an egg in it, and then seal the groove with the secretions of the oviduct. According to the existing literature reports on the method of plant tissue frozen section, it is impossible to prepare complete and good frozen sections of the egg chambers of Anoplophora glabripennis on Elaeagnus angustifolia. This is because the wood of Elaeagnus angustifolia is dense and hard, the tree tissues in the infected area are very fragile and easy to separate and break, and the eggs are fragile and easy to break. After embedding and fixing, the sectioning effect is not ideal, the staining effect is poor, and the tree tissues are broken and lose integrity. Analyzing the reasons, it may be related to factors such as the damage of tree tissues near the egg chambers of Anoplophora glabripennis on Elaeagnus angustifolia, the thickening of the cambium cell wall, gum secretion, the formation of gum ducts, and the extrusion of egg masses. These structures have different morphologies under frozen sections of different thicknesses. In addition, the degree of lignification in the tissues near the egg chambers of Anoplophora glabripennis on Elaeagnus angustifolia is high, which will cause the tissues to be fragile in the frozen state. At present, the method of frozen section of plant tissues for spatial transcriptomics analysis, especially for woody plants, is not perfect, and there is no mature technical system for preparing frozen sections of plant tissues required for spatial transcriptomics analysis. Summary of the Invention

[0005] A method for preparing frozen sections of tree tissues for spatial transcriptomics analysis, comprising the following steps:

[0006] S1. Sample collection and processing

[0007] Cut a large branch of Elaeagnus angustifolia with fresh galleries of Anoplophora glabripennis, and remove the area from the bark to the secondary xylem of the previous year with galleries. Further divide the wood segment, first split it longitudinally, and then further divide it, paying attention to reducing the number of transverse cuts. Retain the samples that are split into triangles or fans extending from the bark to the inside, ensuring that the position of the egg chamber (target area) is 0.3 cm or more away from the edge of the sample. After visually confirming the presence of eggs, use forceps and scissors pre-cooled at 4°C to cut each sample tissue part to a thickness of about 3 mm. Wash the samples with sterilized ultrapure water, blot dry, and place them in a petri dish.

[0008] S2. Sample fixation and vacuum pumping

[0009] Immerse the egg chamber tissue of Anoplophora glabripennis on Elaeagnus angustifolia in 1 ml of 0.05% Tween 20 at room temperature for 15 min. Pre-cool the 75% OCT embedding agent solution on ice (7.5 ml OCT + 2.5 ml sterilized water, invert and mix well). Add 500 µl of 75% OCT to a 1.5 ml centrifuge tube, immerse the egg chamber tissue of Anoplophora glabripennis on Elaeagnus angustifolia in it, and place a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissue from floating on the surface of the OCT during the vacuum pumping process. Open all tube caps and place them in a vacuum concentrator. Pump vacuum at room temperature for 10 min to promote the full wrapping of the OCT solution and its entry into the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0010] S3. Sample embedding and preservation

[0011] Place the embedding cassette on ice, mark the embedding cassette, add an appropriate amount of OCT embedding agent to the bottom of the embedding cassette, about 1 / 4 depth. After slight solidification, put the processed egg chamber tissue of Anoplophora glabripennis on Elaeagnus angustifolia into the embedding cassette containing OCT, add pre-cooled OCT until the tissue is completely wrapped, and adjust the position of the tissue in the OCT to ensure that the target plane of the sample is horizontal with the section plane, and confirm that there are no bubbles near the tissue. Avoid generating bubbles during this process. If bubbles are generated, suck them out with a 200 µl pipette tip. Freeze and solidify the embedding cassette containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0012] S4. Sample sectioning and slide preparation

[0013] Section the sample under the condition that the OCT is completely frozen. Fix the sample holder to the pre-cooled sample holder of the cryostat for sectioning, and the section thickness is 10 μm. Spread the section flat on the quick-freezing table, adjust the angle of the section, and stick the section on a slide dedicated to spatial transcriptomics to obtain a section for spatial transcriptomics analysis of the tree tissue at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia.

[0014] Furthermore, the large branches of Elaeagnus angustifolia in step S1 are large branches of Elaeagnus angustifolia that are 6 years old.

[0015] Furthermore, the OCT embedding agent in steps S2 and S3 is Sakura brand OCT frozen section embedding agent SAKURA, item number 4583.

[0016] Wood tissue sections from the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia prepared by the above-mentioned frozen section method were used for spatial transcriptomics analysis.

[0017] In the present invention, "groove" refers to the grooves made by insects using their mouthparts to gnaw or dig on the surface of trees, and then the ovipositor is inserted into the interior of the tree through the groove to lay eggs; "egg chamber" is the cavity where the eggs are located.

[0018] In the present invention, "pests and diseases" refer to pathogens or insects that damage trees, including but not limited to fungi, bacteria, viruses, mycoplasma, nematodes, and insects that damage trees. In a specific embodiment, the pests and diseases are insects of the family Cerambycidae, preferably Anoplophora glabripennis.

[0019] In the existing technology of frozen section preparation, dehydration and staining are generally performed. Dehydration can be performed directly using a high-concentration sucrose solution, or dehydration can be performed step by step using sucrose solutions with different concentration gradients. This is to obtain a better tissue morphology; staining is to make the tissue structure clearly discernible under a microscope.

[0020] In the present invention, the use of sucrose solution for dehydration will cause the gum in the sample to swell, lose its original tissue morphology, and directly destroy the tissue structure of the wood, making it impossible to slice; staining will cause the structure to be blurred.

[0021] Specifically, the present invention provides the following technical solutions:

[0022] 1. A method for preparing frozen sections of tree tissues damaged by pests and diseases, characterized in that the method comprises the following steps:

[0023] a. Sample processing, obtaining samples containing areas infested by pests and diseases;

[0024] b. Sample fixation, using fixative;

[0025] c. Sample embedding: using OCT embedding medium for embedding;

[0026] d. Slice;

[0027] Wherein, dehydration is not performed in step a.

[0028] 2. The method according to item 1, characterized in that no dyeing is performed in step a and step d.

[0029] 3. The method according to item 1, wherein the sample is a triangular or fan-shaped sample.

[0030] 4. The method according to item 1, wherein the pest-damaged area is not less than 0.3 cm from the edge of the sample, preferably 0.3 - 1 cm, and most preferably 0.3 cm.

[0031] 5. The method according to item 1, wherein the fixing liquid is OCT embedding medium, preferably OCT embedding medium with a volume fraction of 75%.

[0032] 6. The method according to any one of items 1 - 5, wherein step b further includes a vacuum pumping step.

[0033] 7. The method according to any one of items 1 - 6, wherein the frozen section of the pest-damaged tree tissue is a frozen section used for spatial transcriptomics sequencing and analysis.

[0034] 8. The method according to any one of items 1 - 7, wherein the tree tissue is Elaeagnus plant tissue, preferably Elaeagnus angustifolia tissue.

[0035] 9. The method according to any one of items 1 - 8, wherein the pest-damaged tree tissue is the tree tissue damaged by pests within 7 days, preferably within 6 days, preferably within 5 days, preferably within 4 days, preferably within 3 days, preferably within 2 days, and more preferably within 1 day.

[0036] 10. A frozen section of pest-damaged tree tissue, wherein the frozen section of the pest-damaged tree tissue is prepared by the method according to any one of items 1 - 9.

[0037] In some embodiments, the tree tissue can be Elaeagnus plant tissue, preferably the tissue of Elaeagnus angustifolia, Elaeagnus moorcroftii, or Elaeagnus oxycarpa.

[0038] In some embodiments, the tree tissue can also be the tissue of Robinia pseudoacacia, Populus euphratica, Fraxinus spp., Acer spp., Quercus sp., or Populus spp.

[0039] In some embodiments, the tree tissue can also be the tissue of plants of the genus Pinus, Abies, Picea, Tsuga, Pseudotsuga, Larix, or Cedrus.

[0040] Advantages of the present invention:

[0041] The method for preparing frozen sections of tree tissue for spatial transcriptomics analysis provided by the present invention reduces cross-cutting during sample collection and sample segmentation to form a fan-shaped sample. After fixation and evacuation, embedding and sectioning are directly carried out without dehydration and staining. The obtained tree tissue structure after sectioning is complete and the cells are clear, effectively avoiding the loss of intracellular genetic material, which is a frozen sectioning method with good effects. Using tree samples damaged by pests and diseases within 7 days to prepare sections, the tree structure is more complete and clear, and it better meets the requirements of spatial transcriptome sequencing. The present invention is particularly suitable for preparing frozen sections of tree tissue at the oviposition chamber site of Anoplophora glabripennis on Elaeagnus angustifolia in spatial transcriptomics analysis. Description of the drawings

[0042] Figure 1 It is an image of a frozen section of tree tissue at the oviposition chamber site of Anoplophora glabripennis on Elaeagnus angustifolia obtained in Example 1 of the present invention.

[0043] Figure 2 It is an image of a frozen section of tree tissue at the oviposition chamber site of Anoplophora glabripennis on Elaeagnus angustifolia obtained in Comparative Example 4 of the present invention.

[0044] Figure 3 It is an image of a frozen section of tree tissue at the oviposition chamber site of Anoplophora glabripennis on Elaeagnus angustifolia obtained in Comparative Example 6 of the present invention.

[0045] Figure 4 It is an image of a frozen section of tree tissue at the oviposition chamber site of Anoplophora glabripennis on Elaeagnus angustifolia obtained in Comparative Example 7 of the present invention. Detailed implementation manners

[0046] Example 1

[0047] The steps of the method for preparing frozen sections of tree tissue at the oviposition chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptomics analysis are as follows:

[0048] 1. Sample collection and sample segmentation: Collect samples of Elaeagnus angustifolia tree tissues with Anoplophora glabripennis egg chambers. Mark large branches of Elaeagnus angustifolia (branch age 6 years) with fresh Anoplophora glabripennis engrailed grooves formed within 24 hours in the forest, and cut them into 1 m segments. Further segment the wood segments. First, split them longitudinally, and then further segment them, paying attention to reducing the number of transverse cuts. Retain the samples that extend from the bark inward and are segmented into triangles or fans, ensuring that the part where the egg chamber is located (target area) is 0.3 cm or more away from the sample edge. It can be observed with the naked eye whether there are eggs in the Anoplophora glabripennis egg chambers on Elaeagnus angustifolia. After determining that there are eggs, carefully cut each sample tissue part to a thickness of about 3 mm with a knife pre-cooled at 4°C. Wash the surface of the samples with sterilized ultrapure water, dry the surface moisture with dust-free paper, and place them in clean petri dishes.

[0049] 2. Sample fixation and vacuum pumping: Immerse the sample tissues in 1 ml of 0.05% Tween 20 at room temperature for 15 minutes. Pre-cool 75% Sakura brand OCT freezing section embedding medium SAKURA (7.5 ml OCT + 2.5 ml sterilized water, invert and mix well) on ice. Add 500 µl of 75% OCT embedding medium to a 1.5 ml centrifuge tube, immerse the sample tissues in it, and place a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissues from floating on the surface of the OCT during the vacuum pumping process. Open all tube caps and place them in a vacuum concentrator. Pump vacuum at room temperature for 10 minutes to promote the OCT solution to fully wrap and enter the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0050] 3. Embedding and storing samples: Place the embedding cassette on ice, mark the embedding cassette, add an appropriate amount of OCT embedding medium to the bottom of the embedding cassette, about 1 / 4 depth. After slightly solidifying, put the processed sample tissues into the embedding cassette containing OCT, add pre-cooled OCT embedding medium until the tissues are completely wrapped, and adjust the position of the tissues in the OCT to ensure that the target plane of the sample is horizontal with the cutting surface, and confirm that there are no bubbles near the tissues. Bubbles should be avoided during this process. If there are bubbles, they can be sucked out using a pipette. Freeze and solidify the embedding cassette containing the sample tissues and OCT. Wrap the OCT-embedded tissue blocks and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0051] 4. Sample sectioning and slide making: Section the samples in a completely frozen state of OCT. Fix the sample holder to the pre-cooled sample holder of the cryostat for sectioning, and the section thickness is 10 μm. Spread the sections flat on a quick-freezing table, adjust the angle of the sections, stick the sections on the slides dedicated to spatial transcriptomics, observe the section structure of the samples under a microscope, and collect the sections that meet the requirements.

[0052] The imaging of frozen sections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia is as follows Figure 1 as shown. It can be seen from Figure 1 that for the sections prepared by this method, the tree tissue structure obtained after sectioning is complete, the cells are clear, and the loss of intracellular genetic material is effectively avoided. This is a freezing sectioning method with good results and is suitable for the freezing sectioning operation of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia in spatial transcriptomics analysis.

[0053] Comparative examples

[0054] Comparative example 1: The sectioning method is as described in Example 1, except that the step of dehydration with 35% sucrose is added.

[0055] Comparative example 2: The sectioning method is as described in Example 1, except that the step of staining is added.

[0056] Comparative example 3: The sectioning method is as described in Example 1, except that no vacuum is pumped during the fixation process.

[0057] Comparative example 4: The sectioning method is as described in Example 1, except that the sample shape is rectangular.

[0058] Comparative example 5: The sectioning method is as described in Example 1, except that the thickness of the sample is 5 mm.

[0059] Comparative example 6: The sectioning method is as described in Example 1, except that the position of the egg chamber (target area) is 0.1 cm from the edge of the sample.

[0060] Comparative example 7: The sectioning method is as described in Example 1, except that samples with old grooves (for example, the grooves are formed for more than 7 days) are used.

[0061] The sectioning results using different methods are shown in Table 1.

[0062] Table 1

[0063]

[0064] Specifically, the steps of Comparative example 1 are as follows

[0065] The method for preparing frozen sections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptomics analysis is as follows

[0066] 1. Sample collection and sample segmentation: Collect tree tissue samples from the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia, mark the fresh Anoplophora glabripennis grooves formed within 24 hours in the forest, cut off the large branches of Elaeagnus angustifolia (6 years old) with an electric saw, and cut them into 1m sections. Further segment the wood sections, first split them longitudinally, and then further segment them into fan-shaped samples, ensuring that the egg chamber is more than 0.3 cm away from the edge of the sample. Wash the sample with sterilized ultrapure water, immerse it in 35% sucrose solution, and place it in a 4℃ refrigerator for dehydration for 10 hours. The gum in the sample swells and loses its integrity.

[0067] It can be seen from the frozen sections of the tree tissues at the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia used for spatial transcriptome analysis that taking fresh samples of Anoplophora glabripennis with egg chambers and grooved parts on large branches of Elaeagnus angustifolia and soaking them in 35% sucrose solution will cause the gum in the sample to swell and lose its original tissue morphology, directly destroying the tree tissue structure, making it impossible to slice, and unable to be used as the frozen sections of the tree tissues at the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia in spatial transcriptome analysis. Compared with Comparative Example 1, Example 1 can obtain frozen sections of tree tissues at the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia suitable for spatial transcriptome analysis.

[0068] Specifically, the steps of Comparative Example 2 are as follows:

[0069] The steps for preparing frozen sections of tree tissue from the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptome analysis are as follows:

[0070] 1. Sample collection and sample segmentation: Collect tree tissue samples from the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia, mark the fresh Anoplophora glabripennis grooves formed within 24 hours in the forest, cut off the large branches of Elaeagnus angustifolia (6 years old) with an electric saw, and cut them into 1m sections. Further segment the wood sections, first split them longitudinally, and then further segment them, pay attention to reducing the number of transverse cuts, retain the samples extending from the bark to the inside, and segment them into fan-shaped sections, and ensure that the egg chamber is more than 0.3 cm away from the edge of the sample. The naked eye can observe whether there are eggs in the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia. After confirming that there are eggs, use a knife pre-cooled at 4℃ to carefully slice each sample tissue part to a thickness of about 3 mm. Wash the surface of the sample with sterilized ultrapure water, dry the surface moisture with dust-free paper, and place it in a clean culture dish. Use the "Safranin-Fast Green" method for staining.

[0071] 2. Sample fixation and vacuum pumping: Immerse the sample tissue in 1 ml of 0.05% Tween 20 for 15 min at room temperature. Pre-cool 75% SAKURA OCT compound (SAKURA, 7.5 ml OCT + 2.5 ml sterilized water, invert and mix well) on ice. Add 500 µl of 75% OCT compound to a 1.5 ml centrifuge tube, immerse the sample tissue in it, and put a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissue from floating on the surface of OCT during vacuum pumping. Open all tube caps and place them in a vacuum concentrator. Pump vacuum at room temperature for 10 min to promote the full wrapping of the OCT solution and its entry into the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0072] 3. Embedding and storing the sample: Place the embedding cassette on ice, label the embedding cassette, add an appropriate amount of OCT compound to the bottom of the embedding cassette, about 1 / 4 depth. After slightly solidifying, put the processed sample tissue into the embedding cassette containing OCT, add pre-cooled OCT compound until the tissue is completely wrapped, and adjust the position of the tissue in the OCT to ensure that the target plane is horizontal with the section plane, and confirm that there are no bubbles near the tissue. Bubbles should be avoided during this process. If there are bubbles, they can be aspirated using a pipette. Freeze and solidify the embedding cassette containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0073] 4. Sample sectioning and slide preparation: Section the sample under the condition that the OCT is completely frozen. Fix the sample holder to the pre-cooled sample holder of the cryostat and section the sample with a thickness of 10 μm. Spread the section flat on the quick-freezing table, adjust the angle of the section, and attach the section to the slide dedicated to spatial transcriptomics. Observe the section structure of the sample under the microscope.

[0074] For the frozen sections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptomics analysis, it can be seen that when taking the part of the large branch of Elaeagnus angustifolia with fresh egg chambers and grooved parts of Anoplophora glabripennis, and then staining using the "safranin-fast green" method, the tree structure becomes blurred and cannot be used as the operation of frozen sections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia in spatial transcriptomics analysis. Compared with Comparative Example 2, Example 1 can obtain frozen sections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia suitable for spatial transcriptomics analysis.

[0075] Specifically, the steps of Comparative Example 3 are as follows:

[0076] The preparation method steps of frozen sections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptomics analysis are as follows:

[0077] 1. Sample collection and sample segmentation: Collect tree tissue samples from the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia, mark fresh Anoplophora glabripennis grooves formed within 24 hours in the forest, cut off the large branches of Elaeagnus angustifolia (6 years old) with an electric saw, and cut them into 1m sections. Further segment the wood sections, first split them longitudinally, and then further segment them, paying attention to reducing the number of transverse cuts, retaining the samples extending from the bark to the inside, and segmenting them into fan-shaped sections, and ensuring that the egg chamber is more than 0.3 cm away from the edge of the sample. The naked eye can observe whether there are eggs in the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia. After confirming that there are eggs, use a knife pre-cooled at 4℃ to carefully slice each sample tissue part to a thickness of about 3 mm. Wash the surface of the sample with sterilized ultrapure water, dry the surface moisture with dust-free paper, and place it in a clean culture dish.

[0078] 2. Sample fixation: Soak the sample tissue in 1 ml 0.05% Tween 20 at room temperature for 15 min. Pre-cool 75% Sakura brand OCT cryosection embedding medium SAKURA (7.5 ml OCT + 2.5 ml sterile water) on ice and mix by inversion. Add 500 µl 75% OCT embedding medium to a 1.5 ml centrifuge tube and soak the sample tissue in it.

[0079] 3. Embedding and preservation of samples: Place the embedding box on ice, mark the embedding box, add an appropriate amount of OCT embedding agent to the bottom of the embedding box, about 1 / 4 depth, and place the processed sample tissue into the embedding box containing OCT after micro-solidification. Add pre-cooled OCT embedding agent until the tissue is completely wrapped, and adjust the position of the tissue in OCT to ensure that the sample target plane is level with the section surface, and confirm that there are no bubbles near the tissue. Avoid bubbles during this process. If there are bubbles, use a pipette to suck them out. Freeze and solidify the embedding box containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and embedding box with tin foil and store directly in a sealed container at -80°C.

[0080] 4. Sample slicing and slide preparation: Slice the sample when the OCT is completely frozen, fix the sample holder to the pre-cooled sample holder of the cryostat, and slice it with a thickness of 10 μm. Spread the slice flat on the quick-freezing table, adjust the slice angle, stick the slice on the slide dedicated to the spatial transcriptome, and observe the sample slice structure under a microscope.

[0081] For the cryosection of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptome analysis, it can be seen that after fixing the part of the large branch of Elaeagnus angustifolia with fresh egg chambers and grooving of Anoplophora glabripennis and directly embedding and sectioning, the obtained tree tissues after sectioning are fragmented, the cells are blurred, there are many bubbles, and the sections are blurred, which is not suitable for the cryosection operation of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia in spatial transcriptomics analysis. Compared with Comparative Example 3, Example 1 can obtain cryosections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia suitable for spatial transcriptomics analysis.

[0082] Specifically, the steps of Comparative Example 4 are as follows:

[0083] The steps for preparing cryosections of tree tissues at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptome analysis are as follows:

[0084] 1. Sample collection and sample segmentation: Collect tree tissue samples at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia. Mark the fresh Anoplophora glabripennis grooving formed within 24 h in the forest. Use a saw to cut off the large branch of Elaeagnus angustifolia (branch age 6 years) and cut it into 1 m segments. Further segment the wood segments. First split them longitudinally and then further divide them into small rectangular samples. It can be observed with the naked eye whether there are eggs in the egg chambers of Anoplophora glabripennis on Elaeagnus angustifolia. After determining that there are eggs, carefully cut each sample tissue part to a thickness of about 3 mm with a knife pre-cooled at 4°C. Wash the samples with sterilized ultrapure water, dry the surface moisture with lint-free paper, and place them in a clean petri dish.

[0085] 2. Sample fixation and vacuum pumping: Immerse the sample tissues in 1 ml of 0.05% Tween 20 at room temperature for 15 min. Pre-cool 75% Sakura brand OCT cryosection embedding medium SAKURA (7.5 ml OCT + 2.5 ml sterilized water, invert and mix well) on ice. Add 500 µl of 75% OCT embedding medium to a 1.5 ml centrifuge tube, immerse the sample tissues in it, and put a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissues from floating on the surface of OCT during the vacuum pumping process. Open all tube caps and place them in a vacuum concentrator. Pump vacuum at room temperature for 10 min to promote the full wrapping of the OCT solution and its entry into the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0086] 3. Embedding and preserving samples: Place the embedding cassette on ice, label the cassette, add an appropriate amount of OCT embedding medium to the bottom of the cassette, about 1 / 4 depth. After slight solidification, place the processed sample tissue into the embedding cassette containing OCT, add pre-cooled OCT embedding medium until the tissue is completely wrapped, and adjust the position of the tissue in the OCT to ensure that the target plane of the sample is horizontal with the section plane. Confirm that there are no air bubbles near the tissue. Avoid generating air bubbles during this process. If there are air bubbles, use a pipette to suck out the air bubbles. Freeze and solidify the embedding cassette containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0087] 4. Sample sectioning and slide preparation: Section the sample while the OCT is completely frozen. Fix the sample holder to the pre-cooled sample holder of the cryostat and section the sample with a thickness of 10 μm. Spread the section flat on a quick-freezing table, adjust the angle of the section, and attach the section to a glass slide dedicated to spatial transcriptomics. Observe the section structure of the sample under a microscope.

[0088] The imaging of the frozen section of the tree tissue at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia is as Figure 2 shown. As Figure 2 can be seen, after the sample is cut into rectangles for embedding and sectioning, the tree tissue structure obtained after sectioning is fragmented and deformed, which is not suitable for the frozen section operation of the tree tissue at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia in spatial transcriptomics analysis. Compared with Comparative Example 4, Example 1 can obtain a frozen section of the tree tissue at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia suitable for spatial transcriptomics analysis.

[0089] Specifically, the steps of Comparative Example 5 are as follows:

[0090] The preparation method of the frozen section of the tree tissue at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptome analysis is as follows:

[0091] 1. Sample collection and sample segmentation: Collect the tree tissue samples at the egg chamber site of Anoplophora glabripennis on Elaeagnus angustifolia. Mark the fresh Anoplophora glabripennis grooves formed within 24 hours in the forest, and use a saw to cut off the large branch of Elaeagnus angustifolia (branch age 6 years) and cut it into 1 m segments. Further divide the wood segment, first split it longitudinally, and then further divide it, paying attention to reducing the number of transverse cuts. Retain the samples that extend from the bark to the inside and are divided into fan shapes, ensuring that the position of the egg chamber is more than 0.3 cm away from the sample edge. Visually observe whether there are eggs in the egg chamber of Anoplophora glabripennis on Elaeagnus angustifolia. After determining that there are eggs, carefully cut each sample tissue part to a thickness of about 5 mm with a knife pre-cooled at 4°C. Wash the surface of the sample with sterilized ultrapure water, dry the surface moisture with lint-free paper, and place it in a clean petri dish.

[0092] 2. Sample fixation and vacuum pumping: Immerse the sample tissue in 1 ml of 0.05% Tween 20 for 15 min at room temperature. Pre-cool 75% SAKURA OCT compound (SAKURA, 7.5 ml OCT + 2.5 ml sterilized water, invert and mix well) on ice. Add 500 µl of 75% OCT compound to a 1.5 ml centrifuge tube, immerse the sample tissue in it, and place a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissue from floating on the surface of the OCT during vacuum pumping. Open all tube caps and place them in a vacuum concentrator. Pump vacuum at room temperature for 10 min to promote the OCT solution to fully wrap and enter the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0093] 3. Embedding and storing the sample: Place the embedding cassette on ice, mark the embedding cassette, add an appropriate amount of OCT compound to the bottom of the embedding cassette, about 1 / 4 depth. After slightly solidifying, place the processed sample tissue into the embedding cassette containing OCT, add pre-cooled OCT compound until the tissue is completely wrapped, and adjust the position of the tissue in the OCT to ensure that the target plane of the sample is horizontal with the section plane. Confirm that there are no air bubbles near the tissue. Since the sample is thick, it is difficult to avoid generating air bubbles during this process. Try to suck out the air bubbles with a pipette. Freeze and solidify the embedding cassette containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0094] 4. Sample sectioning and slide preparation: Section the sample in a completely frozen state of OCT. Fix the sample holder to the pre-cooled sample holder of the cryostat and section the sample with a thickness of 10 μm. Spread the section flat on a quick-freezing table, adjust the angle of the section, attach the section to a slide dedicated for spatial transcriptomics, observe the section structure of the sample under a microscope, and collect the sections that meet the requirements.

[0095] It can be seen from the frozen tissue sections prepared from the tree sample of the egg chamber part of Anoplophora glabripennis on Elaeagnus angustifolia with a thickness of 5 mm that the part of the large branch of Elaeagnus angustifolia with fresh egg chambers and grooving parts of Anoplophora glabripennis was fixed, vacuum-pumped, directly embedded and sectioned. However, due to the excessive thickness of the sample, the embedding effect was not ideal, and the obtained tree tissue structure after sectioning was incomplete. Therefore, the tissue structure of the frozen sections obtained in Example 1 (sample thickness 2 - 4 mm) is more complete than that in Example 5.

[0096] Specifically, the steps of Comparative Example 6 are as follows:

[0097] The preparation method steps of the frozen tissue sections of the tree tissue of the egg chamber part of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptomics analysis are as follows:

[0098] 1. Sample collection and sample segmentation: Collect tree tissue samples from the egg chamber area of Anoplophora glabripennis on Elaeagnus angustifolia. Mark the fresh A. glabripennis galleries formed within 24 hours in the forest. Use a saw to cut off a large branch (6-year-old branch) of E. angustifolia and cut it into 1 m segments. Further segment the wood segments. First, split them longitudinally and then further segment, paying attention to reducing the number of transverse cuts. Retain the samples that extend from the bark inward and are segmented into fan shapes, ensuring that the location of the egg chamber (target area) is 0.1 cm away from the sample edge. It can be observed with the naked eye whether there are eggs in the egg chamber of A. glabripennis on E. angustifolia. After determining that there are eggs, carefully cut each sample tissue part to a thickness of about 3 mm with a knife pre-cooled at 4°C. Wash the sample surface with sterile ultrapure water, blot the surface moisture with lint-free paper, and place it in a clean petri dish.

[0099] 2. Sample fixation and vacuum pumping: Immerse the sample tissue in 1 ml of 0.05% Tween 20 at room temperature for 15 min. Pre-cool 75% Sakura OCT compound embedding medium SAKURA (7.5 ml OCT + 2.5 ml sterile water, invert and mix well) on ice. Add 500 µl of 75% OCT embedding medium to a 1.5 ml centrifuge tube, immerse the sample tissue in it, and place a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissue from floating on the surface of the OCT during the vacuum pumping process. Open all tube caps and place them in a vacuum concentrator. Pump vacuum at room temperature for 10 min to promote the full wrapping of the OCT solution and its entry into the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0100] 3. Embedding and storing samples: Place the embedding cassette on ice, mark the embedding cassette, add an appropriate amount of OCT embedding medium to the bottom of the embedding cassette, about 1 / 4 depth. After slight solidification, put the processed sample tissue into the embedding cassette containing OCT, add pre-cooled OCT embedding medium until the tissue is completely wrapped, and adjust the position of the tissue in the OCT to ensure that the target plane of the sample is horizontal with the cutting surface, and confirm that there are no bubbles near the tissue. Bubbles should be avoided during this process. If there are bubbles, they can be sucked out using a pipette. Freeze and solidify the embedding cassette containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0101] 4. Sample sectioning and slide making: Section the sample under the fully frozen state of OCT. Fix the sample holder to the pre-cooled sample holder of the cryostat and section, with a section thickness of 10 µm. Spread the sections flat on a quick-freezing table, adjust the angle of the sections, stick the sections on the slides dedicated for spatial transcriptomics, and observe the section structure of the samples under a microscope to collect the sections that meet the requirements.

[0102] The imaging of the frozen sections of the tree tissue at the egg chamber area of A. glabripennis on E. angustifolia is as Figure 3As shown in Figure 3 From the tree tissue sections of the egg chambers of Anoplophora glabripennis on Elaeagnus angustifolia, it can be seen that fresh egg chambers and grooved parts of Anoplophora glabripennis on large branches of Elaeagnus angustifolia were fixed and evacuated, and then directly embedded and sectioned. Since the position of the egg chamber (target area) is too close to the edge of the sample, it is easy to break during the sectioning process, resulting in incomplete sections and incomplete tree tissue structures obtained after sectioning. Therefore, the tissue structure of the frozen sections obtained in Example 1 is clearer than that in Example 2.

[0103] Specifically, the steps of Comparative Example 7 are as follows:

[0104] The method for preparing frozen sections of tree tissues at the egg chamber sites of Anoplophora glabripennis on Elaeagnus angustifolia for spatial transcriptome analysis is as follows:

[0105] 1. Sample collection and sample segmentation: Collect tree tissue samples at the egg chamber sites of Anoplophora glabripennis on Elaeagnus angustifolia. Mark the grooved parts of Anoplophora glabripennis that have formed for more than 7 days (e.g., 8 days) in the forest. Use a saw to cut off the large branches of Elaeagnus angustifolia (branch age 6 years) and cut them into 1 m segments. Further divide the wood segments. First, split them longitudinally and then further divide them, paying attention to reducing the number of transverse cuts. Retain the samples that extend from the bark to the inside and are segmented into fan shapes. Carefully cut each sample tissue part to a thickness of about 3 mm with a knife pre-cooled at 4°C. Wash the surface of the samples with sterile ultrapure water, dry the surface moisture with dust-free paper, and place them in a clean petri dish.

[0106] 2. Sample fixation and evacuation: Immerse the sample tissues in 1 ml of 0.05% Tween 20 at room temperature for 15 min. Pre-cool 75% Sakura brand OCT frozen section embedding medium SAKURA on ice (7.5 ml OCT + 2.5 ml sterile water, invert and mix well). Add 500 µl of 75% OCT embedding medium to a 1.5 ml centrifuge tube, immerse the sample tissues in it, and place a disposable sterile 200 µl pipette tip into the 1.5 ml centrifuge tube to prevent the tissues from floating on the surface of the OCT during the evacuation process. Open all tube caps and place them in a vacuum concentrator. Evacuate at room temperature for 10 min to promote the full wrapping of the OCT solution and its entry into the tissue voids, ensuring the integrity of the OCT-filled embedded tissue.

[0107] 3. Embedding and preserving samples: Place the embedding cassette on ice, label the cassette, add an appropriate amount of OCT embedding medium to the bottom of the cassette, about 1 / 4 depth. After slight solidification, place the processed sample tissue into the embedding cassette containing OCT, add pre-cooled OCT embedding medium until the tissue is completely wrapped, and adjust the position of the tissue in the OCT to ensure that the target plane of the sample is horizontal with the section plane. Confirm that there are no air bubbles near the tissue. Bubbles should be avoided during this process. If there are air bubbles, a pipette can be used to suck them out. Freeze and solidify the embedding cassette containing the sample tissue and OCT. Wrap the OCT-embedded tissue block and the embedding cassette with tin foil and directly store them in a sealed container at -80°C.

[0108] 4. Sample sectioning and slide preparation: Section the sample in a completely frozen state of OCT. Fix the sample holder to the pre-cooled sample holder of the cryostat and section the sample with a thickness of 10 μm. Lay the section flat on the quick-freezing table, adjust the angle of the section, attach the section to a slide dedicated for spatial transcriptomics, observe the section structure of the sample under a microscope, and collect the sections that meet the requirements.

[0109] Combined with Figure 4 As shown, in the old galleries of Anoplophora glabripennis on Elaeagnus angustifolia (for example, the galleries formed more than 7 days ago), the tree tissues in the galleries healed and ruptured, the tree tissue structure was broken and deformed, and the cavity area was filled with tree gum, and the sections did not meet the requirements for spatial transcriptomics sequencing.

[0110] Therefore, in contrast, using tree samples damaged by pests and diseases within 7 days to prepare sections, the tree structure is more complete and clear, and more meets the requirements for spatial transcriptomics sequencing.

[0111] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various transformations can be made to the technical solutions of the present invention, and these simple variants all belong to the protection scope of the present invention.

[0112] In addition, it should be noted that, in the above specific embodiments, the various specific technical features and steps described can be combined in any appropriate way without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods. In addition, any combination can be made between different embodiments of the present invention, as long as it does not violate the idea of the present invention, it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for preparing frozen sections of tree tissues damaged by pests and diseases, characterized in that: The method comprises the following steps: a. Sample processing, obtaining samples containing areas infested by pests and diseases; b. Sample fixation, using fixative; c. Sample embedding: using OCT embedding medium for embedding; d. Slice; Wherein, dehydration is not performed in step a.

2. The method according to claim 1, characterized in that No dyeing is performed in step a and step d.

3. The method according to claim 1, characterized in that The sample is a triangular or fan-shaped sample.

4. The method according to claim 1, characterized in that The distance between the pest-infested area and the edge of the sample is not less than 0.3 cm, preferably 0.3-1 cm, and most preferably 0.3 cm.

5. The method according to claim 1, characterized in that The fixing liquid is an OCT embedding agent, preferably an OCT embedding agent with a volume fraction of 75%.

6. The method according to any one of claims 1 to 5, characterized in that The step b also includes a vacuuming step.

7. The method according to any one of claims 1 to 6, characterized in that The frozen sections of tree tissues infested by pests and diseases are frozen sections used in spatial transcriptomics sequencing and analysis.

8. The method according to any one of claims 1 to 7, characterized in that The tree tissue is Elaeagnus plant tissue, preferably Elaeagnus angustifolia tissue.

9. The method according to any one of claims 1 to 8, characterized in that The tree tissue attacked by pests and diseases is tree tissue attacked by pests and diseases within 7 days, preferably tree tissue attacked by pests and diseases within 6 days, preferably tree tissue attacked by pests and diseases within 5 days, preferably tree tissue attacked by pests and diseases within 4 days, preferably tree tissue attacked by pests and diseases within 3 days, preferably tree tissue attacked by pests and diseases within 2 days, and more preferably tree tissue attacked by pests and diseases within 1 day.

10. Cryosections of tree tissues infested by pests and diseases, characterized in that: The frozen sections of tree tissues infested by pests and diseases are prepared by the method according to any one of claims 1 to 9.

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