Preparation method of insect tissue frozen section with chitin shell

Abstract

The invention provides the preparation method of the insect tissue frozen section with the chitin shell, the chitin shell of the insect tissue is softened by using glycerol, the problem that a complete tissue section cannot be obtained due to too hard chitin shell is avoided, and compared with a softening agent Diaphanol, the glycerol softening time is short, the operation is simple, and the cost is low. Not only can the completeness of the obtained tissue and cell morphology be ensured, but also the completeness of transcripts can be ensured. Meanwhile, gas in the tissue is exhausted as much as possible by utilizing a vacuumizing method, so that the completeness of the tissue, cellular morphology and transcripts in the tissue slice is further ensured. The insect tissue frozen section with the chitin shell, which is prepared by the method disclosed by the invention, can be applied to various detection and analysis scenes such as HE staining, RNA-FISH (Ribonucleic Acid-FISH), space transcriptome and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to a method for preparing frozen sections of insect tissues with chitinous shells. Background Art

[0002] Spatial transcriptomics is a technology that analyzes and describes the gene expression profile of specific cell types in spatial dimensions to understand the differences in gene expression between organs, tissues and pathological states. It can analyze the gene expression of genes in different spatial locations of tissues. While obtaining gene expression data, spatial transcriptomics will also map the gene expression data onto tissue microscopic images to obtain gene expression at specific locations of the tissue. This not only retains the spatial location information of the sample, but also obtains the gene expression dynamics of organs or tissues, allowing researchers to discover more valuable things.

[0003] Fluorescence In Situ Hybridization (RNA-FISH) is a method used to study the localization and expression level of RNA in cells. This method uses a fluorescently labeled probe complementary to the target RNA to hybridize with the target RNA molecule in the cell, and determines the expression level and location of the target RNA in the cell by observing the intensity and distribution of the fluorescence signal under a confocal microscope. The RNA-FISH technology of cells has the advantages of short experimental cycle, safety and pollution-free, good probe stability, high sensitivity, economical and affordable, and can use multiple colors to distinguish multiple targets in the same cell. RNA-FISH technology provides a powerful scientific research tool for studying the function of RNA, and has played an important role in genetic diagnosis, tumor genetics, viral infection analysis, etc.

[0004] Cells are the basic units of organisms. The spatial location information of cells is particularly important for studying and understanding the mechanisms of cell biology, tumor biology, developmental biology and other disciplines. Spatial transcriptome and cell RNA fluorescence in situ hybridization techniques have been widely used in the study of organ development, the pathogenesis of cancer and tumors, and the development of new drugs for diseases, and have achieved a series of breakthrough results. However, the application of spatial transcriptome and RNA-FISH technology in insect tissues is relatively lagging, which is related to the specific tissue structure of insect tissues themselves and the complex diversity of tissues. At present, insect tissue sections are mostly used to observe tissue structure, and do not consider the preservation of biological information inside insect tissues. Often after the sectioning is completed, the changes in biological activity such as transcriptional metabolism inside the cell will be lost, which is not suitable for the development of spatial transcriptome and cell RNA fluorescence in situ hybridization technology.

[0005] Compared with animal tissues, insect tissues have a hard chitin shell on the outside. In the process of preparing slices, if the chitin shell is removed by dissection, it is difficult to ensure the integrity of the tissue in situ during the removal of the chitin shell because the insect tissue is very small. If the chitin shell is not removed, the chitin shell and the soft tissue inside have different hardness, which will cause the integrity of the slice to be reduced, which is not conducive to subsequent experimental operations. At present, insect tissue slices include semi-thin slices, paraffin slices, frozen slices and other technologies, among which the frozen slice preparation cycle is short, the damage to the sample is small, and the true state of the insect tissue can be reflected to the greatest extent. The frozen slice method can perform microscopic imaging of insect tissues, and can preserve the biological activities such as transcription and metabolism of cells inside insect tissues, which is conducive to RNA-FISH and spatial transcriptome research. When conducting RNA-FISH and spatial transcriptome experiments, the preparation of frozen slices is particularly important. The tissue structure of the slices and the integrity of RNA directly determine the accuracy and quality of RNA-FISH and spatial transcriptome data. In the prior art, when preparing frozen sections containing chitin shells, the softener Diaphanol is mainly used to soften the chitin shells of insects. Diaphanol is a solution of chlorine dioxide and glacial acetic acid. Its function is to bleach, fade and transparent chitin shells, but the softener takes 3 to 4 days to soften the chitin shells of insects; and in order to prevent leakage of chlorine oxygen, etc. during the softening process, dark brown frosted glass bottles should be used in the dark, and vaseline should be applied to the bottle stoppers. After softening, they need to be washed with alcohol of different concentrations, and because the softener is acidic, sodium thiosulfate and sodium nitrate solutions are also needed to neutralize the acidic softener. The softening process of the softener Diaphanol is relatively cumbersome, the softening time is relatively long, and alcohol and neutralizing agents are required after softening, which will cause RNA molecules in cells to degrade, which is not conducive to subsequent RNA-FISH and spatial transcriptome experiments. The existence of these problems creates certain challenges for the application of RNA-FISH and spatial transcriptome in the study of insect tissue cells with chitin shells. Therefore, there is an urgent need to provide a cryosection method that ensures the tissue and transcript integrity in cryosections of insect tissues with chitinous coatings. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a method for preparing frozen sections of insect tissue with chitinous shells.

[0007] The second object of the present invention is to provide the application of the insect tissue frozen sections obtained by the above preparation method in HE staining, RNA-FISH or spatial transcriptomics detection and analysis.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The invention provides a method for preparing frozen sections of insect tissues with chitinous shells, which comprises the following steps in sequence: obtaining insect tissue samples containing chitinous shells, dehydrating, softening chitin, embedding, and slicing; the softened chitin adopts a glycerol solution; and the concentration of the glycerol solution is 10% to 50%.

[0010] Since insect tissues are relatively small and have a chitinous shell on the outside, the hardness of the chitinous shell and the soft tissue inside are different, which will reduce the continuity of the frozen section tissue and make it easier to cause wrinkles and breakage of the tissue. The present invention finds that the insect tissue containing chitinous shells after low-temperature dissection and dehydration is softened with glycerol and then embedded and sliced, which can well ensure the integrity and clear structure of the insect tissue, and also ensure the integrity of cell morphology and gene transcripts. The tissue sections prepared by the method of the present invention can be used not only for HE staining and RNA-FISH, but also for spatial transcriptome experiments.

[0011] Furthermore, fresh tissue is obtained from living insects at a low temperature of 0 to 8°C.

[0012] Furthermore, the cryogenically dissected tissue sample is fixed and then dehydrated.

[0013] Furthermore, the fixation is performed using a paraformaldehyde solution.

[0014] Preferably, the concentration of the paraformaldehyde solution is 3-5%.

[0015] Furthermore, the dehydration is carried out by sequentially using 14% to 16% and 28% to 32% sucrose solutions.

[0016] Furthermore, the dehydration is carried out by using 15% and 30% sucrose solutions in sequence. The present invention uses a certain concentration of sucrose solution to treat insect tissue with chitinous shells, which can play a dehydrating role and reduce the generation of ice crystals in the insect tissue with chitinous shells during the embedding process.

[0017] Furthermore, the concentration of the glycerol solution is 10% to 20%.

[0018] Preferably, the concentration of the glycerol solution is 15%. The present invention uses a glycerol solution of a certain concentration to soften the chitin of insect tissues with chitin shells, so that the relatively hard and brittle chitin shells can be sliced ​​into complete slices during subsequent frozen sections, avoiding incomplete sections caused by the chitin shell being too hard and the internal tissue being soft, thereby ensuring the integrity of the tissue morphology and the integrity of the transcripts in the tissue sections. The 15% glycerol solution prepared with DEPC water is used to soften the chitin shells of insects, so that the hardness of the chitin shells and the soft tissues are as consistent as possible.

[0019] Furthermore, the treatment time of the glycerol solution is 2 to 4 hours, so as to avoid softening for too long a period of time, which may lead to degradation of transcripts in the tissue, and to avoid softening for too short a period of time, which may be incomplete.

[0020] Furthermore, the embedding is to place the tissue after softening chitin in an embedding agent, evacuate the air, and then perform the embedding operation. Since there are many tracheas in the insect tissue, if there is gas not discharged, the tissue is more likely to break during slicing, so the gas in the tissue is discharged as much as possible by evacuating the air.

[0021] Furthermore, the vacuuming time is 20 to 60 minutes. The present invention places the insect tissue with chitinous shell in an optimal cutting temperature compound (OCT) and vacuumizes for a certain period of time, which can make the OCT fully immersed in the insect tissue with chitinous shell, and the embedding agent and the insect tissue with chitinous shell will be tightly attached together, so as to avoid the insect tissue with chitinous shell being separated from the embedding agent during slicing, resulting in the insect tissue with chitinous shell being unable to be sliced.

[0022] Furthermore, the embedding method is to fix the vacuumed tissue neatly with double-sided tape, and then perform the embedding operation. In the tissue embedding operation, since the insect tissue is very small, it is very time-consuming to discharge one tissue at a time during embedding. Too long a tissue discharge time will reduce the integrity of the transcripts in the tissue. It is also difficult to place as many tissues as possible in the same plane within the shortest possible embedding time, so that as many structurally intact tissues as possible can be captured in one section during slicing for subsequent experiments. Therefore, the antennae tissue is neatly fixed with double-sided tape, and then the tissues fixed with double-sided tape are neatly arranged in layers for embedding, so that more tissues can be obtained per unit area.

[0023] Furthermore, the slice temperature is -18 to -20°C in the slice chamber.

[0024] Furthermore, the sectioning process also includes sticking the sections together and staining.

[0025] Furthermore, the method of sticking the slices is to gently contact the slices with a glass slide treated with poly-lysine at room temperature, so that the slices automatically adhere to the glass slide.

[0026] The present invention optimizes the existing freezing section method operation steps of insect tissue with chitin shell, softens the chitin shell of the insect with a certain concentration of glycerol, and exhausts the air that may exist in the tissue by vacuuming, thereby reducing the separation of the insect tissue with chitin shell and OCT and the fragmentation and fracture of the tissue, ensuring the integrity of the tissue and cell morphology and the integrity of the transcript in the tissue section, and can fix more tissues with double-sided tape, so that as many tissues as possible can be cut per unit area. The freezing section preparation method is suitable for HE staining, RNA-FISH, spatial transcriptome and other experiments.

[0027] Therefore, the present invention provides the use of insect tissue frozen sections obtained by the above method in HE staining, RNA-FISH or spatial transcriptomics detection and analysis.

[0028] Furthermore, the staining includes but is not limited to HE staining.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a method for preparing frozen sections of insect tissue with chitinous shells. The present invention uses glycerol to soften the chitinous shells of insect tissues, thereby avoiding the problem of being unable to obtain complete tissue sections due to the chitinous shells being too hard. Compared with the use of the softener Diaphanol, the time required for glycerol softening is short and the operation process is simple, which can not only ensure that the obtained tissue is complete and the cell morphology is complete, but also ensure the integrity of the transcripts. At the same time, the gas in the tissue can be discharged as much as possible by vacuuming, reducing the separation of the insect tissue with chitinous shells from OCT and the fragmentation and fracture of the tissue during slice formation, further ensuring the integrity of the tissue and cell morphology in the tissue sections and the integrity of the transcripts. The frozen sections of insect tissue with chitinous shells prepared by the method of the present invention can be applied to various detection and analysis scenarios such as HE staining, RNA-FISH and spatial transcriptome detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 For OCT embedding of bee antennal tissue.

[0032] Figure 2 Frozen sections of honey bee antennae softened with Diaphanol and RNA quality testing.

[0033] Figure 3 Softening of frozen sections of bee antennae tissues with glycerol solutions of different concentrations and RNA quality testing.

[0034] Figure 4 RNA-FISH results of the Orco gene in honey bee antennae.

[0035] Figure 5 Spatial transcriptome results for honey bee antennal tissue. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Example 1 Softening of frozen sections of bee antennae tissue by softening agent Diaphanol and glycerol solutions of different concentrations

[0039] 1. Experimental Methods

[0040] 1. Preparation of slices

[0041] (1) Sampling (cryogenic anatomy): Several worker bees are placed in a beehive and anesthetized at 4°C. The third segment of the bee's antennae is gently cut with ophthalmic forceps. The tissue should be kept fresh as quickly as possible to prevent the tissue from being left for too long, which may cause protein and nucleic acid molecules to decompose and denature, leading to cell autolysis and bacterial growth, and failing to reflect the morphological structure of the tissue when it is alive.

[0042] (2) Dehydration: a. Gently remove the antennal tissue with forceps and place it in a 15% sucrose solution prepared with DEPC water for dehydration until the antennal tissue sinks to the bottom of the sucrose solution;

[0043] b. Use forceps to gently remove the antennal tissue and place it in a 30% sucrose solution prepared with DEPC water to continue dehydration until the antennal tissue sinks to the bottom of the sucrose solution.

[0044] (3) Softening chitin: Use forceps to gently remove the antennae and place them in 0%, 10%, 15% and 50% glycerol solutions prepared with Diaphanol or DEPC water to soften the chitin shell of the antennae for 2 hours at room temperature.

[0045] (4) Vacuuming and degassing: Use forceps to gently remove the antennal tissue and place it in OCT (optimal cutting temperature compound) solution. Vacuum the solution in a vacuum device at room temperature for 30 minutes.

[0046] (5) Embedding: Put a double-sided tape in the cell culture dish, gently place the bee antennae on the double-sided tape, and then use tweezers to roll up the double-sided tape from one end to obtain neatly arranged bee antennae tissue. Apply a layer of OCT embedding glue on the sample holder and pre-cool it on dry ice. Place the antennae tissue in the pre-cooled OCT prepared in advance, add another layer of OCT on it, place another layer of bee antennae, and add another layer of OCT on it, so as to completely cover it. This ensures that as many antennae samples as possible are obtained in the section. After the sample holder is placed in dry ice and the OCT solidifies, remove the tissue block, mark it, and place it at -80℃ for later use ( Figure 1 ).

[0047] (6) Sectioning: a. Pre-cool the constant temperature cryostat, adjust the temperature of the sectioning chamber to -20°C, the temperature of the specimen head to -18°C, and replace and install new sections;

[0048] b. Place the sample from -80℃ to -20℃ in advance to balance the sample temperature and stabilize the sample state;

[0049] c. Adjust the angle of the sample, start slicing, and mount it on a lysine-treated slide.

[0050] (7) Adhesion: Gently touch the slice with a glass slide at room temperature. Due to the temperature difference, the slice automatically adheres to the poly-lysine treated glass slide. Remove the glass slide and conduct subsequent experiments such as morphological observation or store at -80℃ for later use.

[0051] 2. HE staining

[0052] HE staining (hematoxylin-eosin staining) is the most commonly used histological staining method, which is used to observe tissue structure, cell morphology, and nuclear chromatin.

[0053] (1) Take the slices out from -80°C and restore them to room temperature, then bake them in a 42°C oven for 40 min;

[0054] (2) The baked slides were washed in PBS solution for 5 min to wash away OCT;

[0055] (3) Immerse the slide in hematoxylin solution for 1 min, rinse with tap water until the surface is clean and light blue appears under a microscope;

[0056] (4) Immerse the slide in 1% hydrochloric acid alcohol for 5 seconds, quickly take it out, rinse it with tap water, and observe the cell nucleus under a microscope until it turns purple-blue;

[0057] (5) 1% ammonia water turns blue, and the cell nucleus turns blue;

[0058] (6) Immerse the slide in eosin staining solution for 30 seconds, rinse with tap water until the surface is clean, and observe under a microscope that the cell nucleus appears blue and the cytoplasm appears red;

[0059] (7) Using gradient alcohol and xylene to make the stained slides transparent can extend the storage time of the slides;

[0060] (8) Seal the slides with neutral gum and observe the images.

[0061] 3. Tissue RNA Extraction and RNA Quality Testing

[0062] (1) Use a microtome to trim and remove the OCT embedding agent around the tissue as much as possible, and cut 20-30 slices for RNA extraction;

[0063] (2) Extract total RNA from tissues using a micro-tissue RNA extraction kit;

[0064] (3) Use the Agilent 2100 Bioanalyzer to accurately and digitally evaluate the integrity (RNA Integrity Number, RIN), purity, or degree of degradation of RNA. The RIN value is a standard for evaluating RNA integrity and intuitively determines the degree of degradation of RNA samples. The RIN value ranges from 1 to 10, and different values ​​represent different degrees of degradation of sample RNA. Generally, the higher the RIN value, the better the RNA quality.

[0065] 2. Experimental Results

[0066] The bee antennae of this embodiment are neatly arranged in OCT and then wrapped as follows. Figure 1 As shown in A, the honey bee antennae tissue was embedded in OCT and then cross-sectioned. Figure 1 As shown in B.

[0067] The bee antennae tissue embedded in the chitin shell softened by Diaphanol is easier to slice. When observing the slices under a microscope, the chitin shell and the soft tissue inside are intact and have no obvious deformation ( Figure 2 A), but the integrity of RNA is poor ( Figure 2 B) It cannot meet the experimental requirements of later RNA-FISH or spatial transcription.

[0068] The results of frozen sections of bee antennae softened by glycerol solutions of different concentrations are as follows Figure 3 As shown in the figure, it is difficult to slice the bee antennae tissue directly embedded in the chitin shell without softening it with glycerol. When observing the slices under a microscope, most of the slices are broken, with multiple tissue separations and severe tissue breakage and shedding. The soft tissue inside is incomplete and has obvious deformation ( Figure 3 A), although the integrity of RNA meets the requirements ( Figure 3B), but due to the poor integrity of the slices, the spatial position of the tissue is severely deformed, which cannot meet the experimental requirements of later RNA-FISH or spatial transcription.

[0069] It is easier to slice under the protection of 10% glycerol, and the quality of the slices is greatly improved, but there is still slight breakage, and only a few of the slices that fail are usable ( Figure 3 C), the integrity of RNA meets the experimental requirements ( Figure 3 D).

[0070] After the bee antennae tissue was softened with 15% glycerol, the integrity of the slices was very good, without damage or wrinkles ( Figure 3 E), the integrity of RNA meets the experimental requirements ( Figure 3 F).

[0071] The quality of honey bee antennal tissue sections softened by 50% glycerol was reduced ( Figure 3 G), it may be that the glycerol concentration in the tissue is too high, making it difficult for OCT to fix the tissue well, and the integrity of RNA meets the experimental requirements ( Figure 3 H).

[0072] Although the chitin shell of the antennae of bees can be softened with the existing Diaphanol softener to ensure a relatively complete slice, the RNA integrity of the tissue softened by Diaphanol is poor, and subsequent RNA-FISH and spatial transcriptome experiments cannot be continued. After the antennae of bees are softened by glycerol, it is easier to prepare slices than the direct slice method, and the overall effect of the slices is improved. The slices are clear and complete, the antennae tissue slices are complete, the tissue is not broken or folded, the cell morphology structure is obvious, and no damage is seen. Considering the integrity of the sliced ​​tissue and the integrity of the gene transcripts, 15% glycerol is selected as a softener for softening the chitin shell of insects, which can keep the samples intact at both macro and micro scales. Chitin softening reagent for insect tissues.

[0073] Example 2 Frozen sections of honey bee antennae and RNA-FISH

[0074] 1. Experimental Methods

[0075] The method for preparing frozen sections is basically the same as that in Example 1, including sampling, dehydration, softening chitin, vacuuming and exhausting gas, embedding, slicing, and sticking. The difference is that:

[0076] 1. After sampling, the tissue is quickly fixed in 4% paraformaldehyde prepared with DEPC water, placed at 4°C overnight, and then dehydrated. Formaldehyde-fixed tissue can be stored at -80°C for a longer time than unfixed tissue without affecting the RNA-FISH experiment, because RNA-FISH only needs to detect RNA molecules in situ in the tissue.

[0077] 2. To soften chitin, use a 15% glycerol solution prepared with DEPC water and soften the chitinous shell of the antennae for 2 hours at room temperature.

[0078] 3. RNA-FISH

[0079] RNA-FISH is a technology that uses fluorescently labeled nucleic acid probes to detect specific RNA sequences in cells. This technology uses fluorescently labeled nucleic acid fragments as probes to complement the RNA to be tested on tissues, cells or chromosomes to form nucleic acid hybrid molecules, and then displays the location of RNA in tissues, cells or chromosomes through a fluorescent detection system.

[0080] 1) Sample hybridization: Prepare a reaction chamber on the tissue section of the sample to be tested, dehydrate and denature the slide after PFA fixation with 100% methanol, digest with protease, add PBST, heat the sample at 65°C for 5 minutes, quickly put it on ice, add hybridization buffer to the reaction chamber and incubate at 37°C overnight;

[0081] 2) Ligation reaction: discard the hybridization solution, wash three times with PBST, add ligase system and react at 25℃ for 3h;

[0082] 3) Rolling circle amplification: discard the ligase reaction system, wash with PBST several times, add the rolling circle amplification reaction system and react at 30°C overnight;

[0083] 4) Fluorescence hybridization: discard the rolling circle amplification reaction system, wash with PBST several times, add the fluorescent probe hybridization system into the above reaction chamber for hybridization in situ color development;

[0084] 5) Use DAPI to stain the cell nucleus: stain with DAPI working solution for 2 minutes at room temperature and rinse twice with PBS;

[0085] 6) After gradient alcohol dehydration, the slides were sealed with neutral gum;

[0086] 7) Microscopic imaging: Image the study area of ​​the sample to be tested and interpret the spatial location information of RNA.

[0087] 2. Experimental Results

[0088] Figure 4The results of RNA-FISH of the Orco gene in the cross-section of bee antennae. The number and distribution of fluorescent signals in the figure can reflect the abundance and location of the target Orco gene in the cell, indicating that the prepared sections can be used for RNA-FISH experiments.

[0089] Example 3 Frozen sections and spatial transcriptome of honey bee antennal tissue cross sections

[0090] 1. Experimental Methods

[0091] The frozen section and RNA-FISH method are basically the same as those in Example 2, including sampling, dehydration, softening chitin, vacuuming to remove gas, embedding, sectioning, and sticking. The difference is that:

[0092] 1. After collecting the sample, proceed directly to the dehydration step. Direct dehydration without formaldehyde fixation can be used for the subsequent experiments, because the spatial transcriptome needs to dissociate RNA molecules, which is more conducive to the capture of RNA molecules.

[0093] 2. Spatial transcriptome

[0094] Spatial transcriptome technology can simultaneously detect the genetic information and spatial location of each cell in the tissue.

[0095] 1) Sample quality inspection: For OCT tissue embedding blocks, perform frozen section HE staining, take photos, and collect multiple frozen section samples for RNA extraction. Subsequent experimental operations can only be performed if the following two prerequisites are met: the sample tissue morphology information is complete and the RNA is not degraded (RIN>=7).

[0096] 2) Tissue permeabilization: Determine the optimal permeabilization time of the sample to ensure the release of RNA in the tissue sample, hybridize with the oligonucleotide probe on the slide for subsequent reverse transcription and synthesis of cDNA.

[0097] 3) Library construction: The library construction process includes a series of experimental processes such as frozen section preparation, ssDNA (HE) staining and photography, permeabilization, cDNA synthesis, cDNA collection into test tubes for PCR amplification, and library construction.

[0098] 4) Sequencing: After the spatial transcriptome library passes the library test, it is sequenced.

[0099] 5) Perform data quality check on sequencing data.

[0100] 2. Experimental Results

[0101] from Figure 5 The experimental results show that the frozen sections of the bee antennae tissue prepared by the method of this embodiment are intact ( Figure 5 A), the sequencing results of spatial transcriptome are as follows Figure 5 As shown in BD, the gene abundance (gene number) and the MID (Molecular indentifer as a molecular tag for gene expression quantification) of the RNA molecule indicate that a high-quality spatial transcriptome library of honeybee antennae was obtained. Therefore, the frozen sections prepared by the method of this embodiment are sufficient to meet the experimental requirements of the spatial transcriptome.

[0102] The results of HE staining, RNA-FISH and spatial transcriptome of honey bee antennae tissues are summarized, indicating that the operation method of the present invention can not only ensure the integrity of tissue and cell morphology during the frozen section process, but also ensure the integrity of transcripts and the quality of the spatial transcriptome library. The setting of each condition in the present invention is reasonable and feasible, and can be used for the preparation of slices of insect tissues with chitinous shells, and the obtained slices can be used for subsequent experimental operations of HE staining, RNA-FISH and spatial transcriptomics.

Claims

1. A method for preparing frozen sections of insect tissue with chitinous shells, characterized in that: The method comprises the following steps: obtaining an insect tissue sample containing a chitin shell, dehydrating, softening the chitin, embedding, and slicing; the softening chitin adopts a glycerol solution; and the concentration of the glycerol solution is 10% to 50%.

2. The preparation method according to claim 1, characterized in that: The obtaining step is to obtain fresh tissue from living insects at a low temperature of 0 to 8°C.

3. The preparation method according to claim 1, characterized in that: The insect tissue samples were fixed and then dehydrated.

4. The preparation method according to claim 3, characterized in that: The fixation is carried out using a paraformaldehyde solution.

5. The preparation method according to claim 1, characterized in that: The dehydration is carried out by sequentially using 14% to 16% and 28% to 32% sucrose solutions.

6. The preparation method according to claim 1, characterized in that: The concentration of the glycerol solution is 10% to 20%.

7. The preparation method according to claim 6, characterized in that: The concentration of the glycerol solution is 15%.

8. The preparation method according to claim 1, characterized in that: The treatment time of the glycerol solution is 2 to 4 hours.

9. The preparation method according to claim 1, characterized in that: The embedding is to place the softened chitinous tissue in an embedding agent, evacuate the vacuum, and then perform the embedding operation.

10. Use of the insect tissue frozen section obtained by the preparation method according to any one of claims 1 to 9 in HE staining, RNA-FISH or spatial transcriptomics detection and analysis.

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