Animal tissue embedding medium for space transcriptomics and space metabonomics analysis and embedding method

Abstract

The invention provides an animal tissue embedding medium for space transcriptomics and space metabonomics analysis and an embedding method, and relates to the technical field of biology, the animal tissue embedding medium comprises carboxymethyl cellulose with the mass concentration of 1.5-2.5%, polyvinylpyrrolidone with the mass concentration of 5.8-7.3% and cane sugar with the mass concentration of 0.5-2.4%, and the animal tissue embedding medium has good fluidity, slice smoothness and tissue supporting performance; a tissue slice prepared from the animal tissue embedding medium can avoid interference peaks in a mass spectrum ionization process, and meanwhile, the animal tissue embedding medium also shows good sequencing quality and relatively high gene capture number in space transcriptome sequencing, and can be suitable for research on space transcriptomics and space metabonomics of animal tissues; therefore, cryopreserved and embedded samples are prevented from being prepared respectively aiming at space transcriptomics and space metabonomics.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an animal tissue embedding agent and an embedding method for spatial transcriptomics and spatial metabolomics analysis. Background Art

[0002] Spatial omics is an emerging field of biomedical research that is dedicated to analyzing the omics information of biological tissues or cells at a spatial scale. Traditional omics technologies, such as transcriptomics, proteomics, and metabolomics, provide us with rich information, but these technologies often lose the spatial information of samples. Spatial omics aims to make up for this shortcoming. It can reveal the heterogeneity of cells and tissues in spatial layout and provide us with a more complete biological perspective. Currently, spatial omics has been widely used in clinical medicine, oncology, pharmacy, environmental toxicology and other fields.

[0003] Spatial transcriptomics (ST) and spatial metabolomics are two cutting-edge omics technologies that provide spatial information on gene expression and metabolite distribution in cells and tissues, respectively. The combination of these two technologies provides a new perspective for studying biology and disease mechanisms. Spatial transcriptomics technology can reveal the gene expression patterns of cells in tissues, while spatial metabolomics can provide information on the distribution of metabolites in tissues. Combining these two technologies, researchers can have a more comprehensive understanding of the structure and function of biological systems at the cellular and molecular levels. For example, by using single-cell sequencing, spatial transcriptome sequencing, and spatial metabolomics technology, spatial multi-omics analysis of brain samples from patients with traumatic brain injury was performed, and a lipid peroxidation area of ​​injured neurons, as well as unbalanced inositol and inositol phosphates and related spatial markers were found. The results highlight the complex transcriptome regulation and metabolic changes in the injured brain and will directly contribute to the design of reagents to target specific genes in the human brain for functional analysis.

[0004] In spatial transcriptomics, the commonly used embedding agent is mainly OCT, the Optimal Cutting Temperature Compound. OCT is a water-soluble freezing medium, which is often used for tissue sections because it can protect the tissue structure during freezing and provide support during sectioning. It is also water-soluble and compatible with a variety of staining procedures. OCT tissue embedding has the following advantages: protecting tissue structure and providing structural support during frozen sections; maintaining the optimal temperature during sectioning to make the sections smooth; being water-soluble and compatible with a variety of staining procedures. However, OCT embedding agents have certain limitations in spatial metabolomics: 1. Potential contamination and ion suppression: OCT-embedded tissues may cause sample contamination and ion suppression problems in mass spectrometry imaging analysis. The polymers in them may generate interference peaks during ion ionization. These interference peaks will mask the true signals of metabolites, especially small molecule metabolites, thereby affecting the detection precision, accuracy and reliability of metabolites, and are not suitable for MALDI-MSI analysis. 2. Preservation issues: For animal tissues with high water content, long-term storage may cause cell dehydration and shrinkage.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide an animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis, so as to solve the technical problem in the prior art that there is a lack of animal tissue embedding agents that can be simultaneously applied to spatial transcriptomics and spatial metabolomics analysis.

[0007] A second object of the present invention is to provide a method for preparing the above-mentioned animal tissue embedding agent.

[0008] The third object of the present invention is to provide the application of the above-mentioned animal tissue embedding agent or the animal tissue embedding agent prepared by the above-mentioned preparation method in the spatial transcriptomics and spatial metabolomics analysis of animal tissues.

[0009] A fourth object of the present invention is to provide an animal tissue freezing embedding method for spatial transcriptomics and spatial metabolomics analysis.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: In a first aspect, the present invention provides an animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis, comprising carboxymethyl cellulose with a mass concentration of 1.5-2.5%, polyvinyl pyrrolidone of 5.8-7.3% and sucrose of 0.5-2.4%.

[0011] Furthermore, it includes 2% carboxymethyl cellulose, 7% polyvinyl pyrrolidone and 1% sucrose in a mass concentration.

[0012] Furthermore, the viscosity of the carboxymethyl cellulose is 1500-3100 mPa.s; The molecular weight of the polyvinyl pyrrolidone is 30,000-40,000.

[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned animal tissue embedding agent, comprising dissolving carboxymethyl cellulose, polyvinyl pyrrolidone and sucrose in pure water according to the formula amount to obtain the animal tissue embedding agent.

[0014] Furthermore, after dissolving, the method further comprises standing for at least 12 hours.

[0015] Furthermore, the dissolving conditions include stirring and dissolving at 60-70°C.

[0016] In a third aspect, the present invention provides the use of the above-mentioned animal tissue embedding agent or the animal tissue embedding agent prepared by the above-mentioned preparation method in the spatial transcriptomics and spatial metabolomics analysis of animal tissues.

[0017] In a fourth aspect, the present invention provides an animal tissue freezing embedding method for spatial transcriptomics and spatial metabolomics analysis, comprising immersing the animal tissue in a pre-cooled animal tissue embedding agent and freezing it to obtain an animal tissue freezing embedding sample; The animal tissue embedding agent is the above-mentioned animal tissue embedding agent or the animal tissue embedding agent prepared by the above-mentioned preparation method.

[0018] Furthermore, the animal tissue is washed with pre-cooled PBS or physiological saline before freezing and embedding.

[0019] Furthermore, the freezing step also includes removing bubbles in the embedding medium.

[0020] The present invention provides an animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis. The animal tissue embedding agent in the range of 1.5-2.5% of carboxymethyl cellulose, 5.8-7.3% of polyvinyl pyrrolidone and 0.5-2.4% of sucrose has good fluidity, section smoothness and tissue support; tissue sections prepared by the embedding agent can avoid interference peaks in the mass spectrometry ionization process, and the animal tissue embedding agent also shows good sequencing quality and a high number of gene captures in spatial transcriptomics sequencing, and can be applied to the study of spatial transcriptomics and spatial metabolomics of animal tissues, so as to avoid the preparation of cryopreserved embedded samples for spatial transcriptomics and spatial metabolomics respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The spatial metabolomics analysis diagram of the frozen embedded sections of mouse intestinal tissue provided in Application Example 2 of the present invention; Figure 2 This is a spatial transcriptomic analysis diagram of frozen embedded sections of mouse intestinal tissue provided in Application Example 2 of the present invention. DETAILED DESCRIPTION

[0023] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0024] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0025] On one hand, the present invention provides an animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis, comprising carboxymethyl cellulose with a mass concentration of 1.5-2.5%, polyvinyl pyrrolidone with a mass concentration of 5.8-7.3%, and sucrose with a mass concentration of 0.5-2.4%.

[0026] Experiments have shown that animal tissue embedding agents in the range of 1.5-2.5% carboxymethyl cellulose, 5.8-7.3% polyvinyl pyrrolidone and 0.5-2.4% sucrose have good fluidity, sectioning smoothness and tissue support; tissue sections prepared by them can avoid interference peaks during mass spectrometry ionization. At the same time, the animal tissue embedding agent also shows good sequencing quality and a high number of gene capture in spatial transcriptome sequencing, which can be used for the study of spatial transcriptomics and spatial metabolomics of animal tissues, so as to avoid the preparation of frozen embedded samples for spatial transcriptomics and spatial metabolomics respectively.

[0027] The mass concentration of the carboxymethyl cellulose may be, but is not limited to, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% or 2.5%, or any concentration between 1.5 and 2.5%.

[0028] The mass concentration of the polyvinyl pyrrolidone may be, but is not limited to, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2% or 7.3%, or any concentration between 5.8% and 7.3%.

[0029] The mass concentration of the sucrose may be, but is not limited to, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2% or 2.4%, or any concentration between 0.5 and 2.4%.

[0030] In some specific embodiments, the animal tissue embedding agent includes 2% carboxymethyl cellulose, 7% polyvinyl pyrrolidone and 1% sucrose in a mass concentration, and has the best fluidity, section smoothness and tissue support performance.

[0031] In some specific embodiments, the viscosity of the carboxymethyl cellulose is 1500-3100 mPa.s, preferably 3100 mPa.s; the molecular weight of the polyvinyl pyrrolidone is 30000-40000, preferably 40000, which meet the viscosity requirements of the animal tissue embedding agent.

[0032] According to another aspect of the present invention, a method for preparing the above-mentioned animal tissue embedding agent is also provided, comprising dissolving carboxymethyl cellulose, polyvinyl pyrrolidone and sucrose in pure water according to the formula amount to obtain the animal tissue embedding agent.

[0033] The animal tissue embedding agent provided by the present invention has a simple preparation process and is convenient for batch production. The prepared animal tissue frozen embedding sample exhibits excellent sectioning effect in frozen sections.

[0034] In some specific embodiments, the dissolution further includes standing for at least 12 hours to remove bubbles in the embedding agent.

[0035] The dissolving conditions include stirring and dissolving at 60-70°C.

[0036] According to another aspect of the present invention, there is also provided the use of the above-mentioned animal tissue embedding agent or the animal tissue embedding agent prepared by the above-mentioned preparation method in the spatial transcriptomics and spatial metabolomics analysis of animal tissues.

[0037] According to another aspect of the present invention, there is also provided an animal tissue freezing embedding method for spatial transcriptomics and spatial metabolomics analysis, comprising immersing the animal tissue in a pre-cooled animal tissue embedding agent for freezing, to obtain an animal tissue frozen embedding sample; the animal tissue embedding agent is the above-mentioned animal tissue embedding agent or the animal tissue embedding agent prepared by the above-mentioned preparation method.

[0038] The animal tissue frozen embedded samples prepared by the animal tissue embedding agent provided by the present invention not only meet the standards of spatial transcriptomics research, but also can effectively reduce the background noise in mass spectrometry detection in spatial metabolomics analysis, thereby ensuring the accuracy of metabolite determination.

[0039] In some specific embodiments, the animal tissue is further washed with pre-cooled PBS or physiological saline before cryo-embedding to remove blood and impurities on the surface of the animal tissue.

[0040] In some specific embodiments, the freezing further comprises removing bubbles in the embedding medium.

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1 An animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis comprises 2% carboxymethyl cellulose, 7% polyvinyl pyrrolidone and 1% sucrose in a mass concentration.

[0043] The viscosity of carboxymethyl cellulose is 3100 mPa.s, and the molecular weight of polyvinyl pyrrolidone is 40,000.

[0044] Example 2 An animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis comprises 1.5% carboxymethyl cellulose, 7.3% polyvinyl pyrrolidone and 2.4% sucrose in mass concentration.

[0045] The viscosity of carboxymethyl cellulose is 1500 mPa.s, and the molecular weight of polyvinyl pyrrolidone is 40000.

[0046] Example 3 An animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis comprises 2.5% carboxymethyl cellulose, 5.8% polyvinyl pyrrolidone and 0.5% sucrose in mass concentration.

[0047] The viscosity of carboxymethyl cellulose is 3100 mPa.s, and the molecular weight of polyvinyl pyrrolidone is 30,000.

[0048] Comparative Example 1 Different from Example 1, the mass concentration of carboxymethyl cellulose is 2.8%.

[0049] Comparative Example 2 Different from Example 1, the mass concentration of carboxymethyl cellulose is 1.2%.

[0050] Comparative Example 3 The difference from Example 1 is that the mass concentration of sucrose is 0.3%.

[0051] Comparative Example 4 The difference from Example 1 is that the mass concentration of sucrose is 2.7%.

[0052] Comparative Example 5 The difference from Example 1 is that the mass concentration of polyvinyl pyrrolidone is 5.5%.

[0053] Comparative Example 6 The difference from Example 1 is that the mass concentration of polyvinyl pyrrolidone is 7.5%.

[0054] Comparative Example 7 The difference from Example 1 is that hydroxypropyl methylcellulose of the same viscosity is used instead of carboxymethyl cellulose.

[0055] Comparative Example 8 Different from Example 1, no carboxymethyl cellulose was added.

[0056] Comparative Example 9 Different from Example 1, no sucrose was added.

[0057] Embodiment 4-6 A method for preparing an animal tissue embedding agent for spatial transcriptomics and spatial metabolomics analysis is prepared according to the formula amounts of Examples 1 to 3, taking 100 ml as an example, according to the following steps: dissolving the weighed carboxymethyl cellulose, polyvinyl pyrrolidone and sucrose in ultrapure water according to the formula amounts, heating and stirring at 60 to 70° C. until the powder is completely dissolved, mixing evenly and letting it stand for 12 hours to remove bubbles.

[0058] Comparative Examples 10~18 The difference from Example 4 is that the preparation is carried out according to the formula amounts of Comparative Examples 1 to 9 respectively.

[0059] Comparative Example 19 The difference from Example 4 is that after heating and stirring at 60-70° C. until the powder is completely dissolved, the mixture is not allowed to stand.

[0060] Comparative Example 20: OCT embedding agent.

[0061] The prepared embedding medium was left to stand for more than 12 hours to observe whether the fluidity of each embedding medium was sufficient to be used as a usable embedding medium. Some mouse tissue samples, including heart, liver, spleen, kidney, brain, testis, and intestine, were further embedded in the prepared different embedding mediums and frozen on dry ice to prepare frozen section samples. The prepared samples were further sliced ​​one by one on a microtome to observe the smoothness of the slices and the effect of the slide spread during the slicing process.

[0062] The following application examples all use mouse intestinal tissue as a sample. The mouse intestinal tissue comes from C57 mice, 10 weeks old, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0063] Application Example 1 The embedding agents of Examples 4 to 6 and Comparative Examples 10 to 20 are used to prepare frozen embedded samples of animal tissues. Specifically, the following steps are followed: the collected mouse small intestinal tissue samples are immediately placed in a pre-cooled PBS solution to wash and remove the blood and intestinal contents on the surface. The sample information and section direction are marked on the embedding box, and the prepared embedding agent is added (the fluidity of the embedding agent of each example is observed during the preparation process), and then the embedding box is placed on ice for pre-cooling, and the tissue sample is placed in the pre-cooled prepared embedding agent to ensure that the tissue is completely covered by the embedding agent and all bubbles are removed. Then the embedding box is placed on dry ice and waits for the embedding agent to solidify. The embedded tissue samples should be stored at -80°C to maintain their structure and RNA integrity.

[0064] Frozen sectioning: Adjust the temperature of the cooling box of the cryostat to -20°C, and set the temperature of the freezing table to between -15°C and -18°C. After the embedded sample obtained in the previous step is placed in the cooling box for 30 minutes, the tissue sample is fixed on the sample tray using OCT. Then, it is placed on the freezing table until the sample is completely bonded to the tray. Next, the sample is moved to the clamping device of the slicer for fixation. After adjusting the slice angle and slice thickness, the slice operation is performed. Select the slices that meet the standards and attach them to the positively charged adhesive slides to observe the smoothness of the slices and the effect of the spread during the slicing process.

[0065] The fluidity evaluation standard is as follows: the fluidity evaluation uses OCT as a control: OCT-like fluidity is 9-10 points, slightly worse fluidity than OCT is 7-8 points, poor fluidity or gel-like is ≤6 points, and the full score is 10 points.

[0066] Evaluation criteria for slice smoothness: Slice smoothness was compared with OCT: slices that were similar to OCT and smooth scored 9 to 10 points, slices that were smooth but had some wrinkles scored 7 to 8 points, slices that were difficult to slice scored ≤ 6 points, with a full score of 10 points.

[0067] Evaluation criteria for the slice spreading effect: The slice spreading effect was compared with OCT: if the cut slices could be easily turned over, moved and flattened with a brush on the exhibition stand, it was scored 9 to 10 points; if the slices were difficult to turn over, move and flatten with a brush on the exhibition stand but did not affect the subsequent patching action, it was scored 7 to 8 points; if the slices were difficult to turn over, move and flatten with a brush on the exhibition stand, and the slices were prone to cracks and breakage, it was scored ≤ 6 points, with a full score of 10 points.

[0068] The specific results are shown in Table 1.

[0069] Table 1 Comparison of sectioning effects of different embedding agents

[0070] As shown in Table 1, compared with Comparative Example 20 (OCT embedding agent), in Example 4, the CMC content is above 2% or the PVP content is above 7% in the embedding agent ratio, which will have a greater impact on the fluidity of the embedding agent, and many bubbles will appear during the configuration process, which is not easy to eliminate even if left for a long time. When the CMC content is below 2% or the PVP content is below 7%, the overall fluidity of the embedding agent will be in a better state, and the fluidity will be better as the content of both decreases, but the slicing effect will be affected, the slices are prone to wrinkles, the overall support is not as good as Example 4, and the edges of the slices are prone to damage and sticking to the brush when the brush is used to spread the slices, which is not conducive to the operation of spreading the slices. When the CMC and PVP contents remain unchanged, the fluidity of the embedding agent can be improved by appropriately increasing the sucrose content. During the embedding agent ratio, more bubbles will appear when heated and stirred. If it is not left to stand overnight, there will be many bubbles in the embedding agent, and cavities will appear in the process of making embedded blocks by dry ice freezing, affecting the slicing.

[0071] Application Example 2: Spatiotemporal transcriptomics and spatial metabolomics analysis of mouse intestine 1. Spatial metabolomics analysis Take the frozen embedded sample prepared by the embedding agent of Example 4 in Application Example 1 and perform 10 μm sectioning. Select the sections that meet the standards and attach them to a positively charged adhesive slide for subsequent spatial metabolomics analysis. AFADESI (Air-flow-assisted desorption electrospray ionization) mass spectrometry imaging technology is used to detect in dual ion mode with a resolution of 50 μm. The specific results are as follows: Figure 1 shown.

[0072] 2. Spatial transcriptomic analysis The frozen embedded sample slices prepared by the embedding agent of Example 4 in Application Example 1 were used for spatial transcriptome analysis. The adjacent slices were attached to the Stereo-seq chip to perform spatial transcriptome analysis. The specific results are as follows: Figure 2 shown.

[0073] Depend on Figure 1 and Figure 2 It can be seen that the sample using the embedding agent of Example 4 can be well applied to spatial transcriptomics and spatial metabolomics. Figure 1 As shown, Figure 1 This is a total ion imaging diagram of the spatial metabolism results of mouse intestinal slices. This figure mainly shows the slice outline and the signal response intensity of each part. The color represents the spatial distribution of the total ion signal of the metabolite molecule in the sample slice. The overall signal intensity distribution is uniform ( Figure 1 The left picture is the result of positive ion mode. Figure 1 The right figure is the negative ion mode result), where the positive ion mode identified more metabolites. Figure 2 As shown in the figure, the sequencing results show that the Clean Ratio of the offline data is 44.23%, and the Clean Reads Q30 result is 91.43%, which can well meet the subsequent analysis. Figure 2 The left figure in the figure shows a complete and clear structure, and the ssDNA staining image ( Figure 2 The structure is accurate and the background is pure. The cell clustering analysis results based on the sequencing data ( Figure 2 The embedding agent provided in Example 4 can provide better support for animal tissue, and has good fluidity and good film spreading effect, and is used for spatial metabolomics analysis, and the background noise generated is small to avoid interference peaks, and the animal tissue embedding agent also shows good sequencing quality and high gene capture number in spatial transcriptome sequencing. Therefore, the embedding agent provided in Example 4 of the present invention can be applied to spatial metabolomics analysis and spatial transcriptomics analysis of animal tissue at the same time.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An animal tissue embedding medium for spatial transcriptomics and spatial metabolomics analysis, characterized in that: It includes carboxymethyl cellulose with a mass concentration of 1.5-2.5%, polyvinyl pyrrolidone with a mass concentration of 5.8-7.3% and sucrose with a mass concentration of 0.5-2.4%.

2. The animal tissue embedding medium according to claim 1, characterized in that: It includes 2% carboxymethyl cellulose, 7% polyvinyl pyrrolidone and 1% sucrose in a mass concentration.

3. The animal tissue embedding medium according to claim 2, characterized in that: The viscosity of the carboxymethyl cellulose is 1500-3100 mPa.s; The molecular weight of the polyvinyl pyrrolidone is 30,000 to 40,000.

4. The method for preparing the animal tissue embedding agent according to any one of claims 1 to 3, characterized in that: The method comprises dissolving carboxymethyl cellulose, polyvinyl pyrrolidone and sucrose in pure water according to a formula to obtain an animal tissue embedding agent.

5. The preparation method according to claim 4, characterized in that: After dissolution, the mixture is allowed to stand for at least 12 hours.

6. The preparation method according to claim 4, characterized in that: The dissolving conditions include stirring and dissolving at 60-70°C.

7. Use of the animal tissue embedding agent according to any one of claims 1 to 3 or the animal tissue embedding agent prepared by the preparation method according to any one of claims 4 to 6 in the spatial transcriptomics and spatial metabolomics analysis of animal tissues.

8. A method for freezing and embedding animal tissues for spatial transcriptomics and spatial metabolomics analysis, characterized in that: The method comprises immersing the animal tissue in a pre-cooled animal tissue embedding medium and freezing it to obtain a frozen embedded animal tissue sample; The animal tissue embedding agent is the animal tissue embedding agent according to any one of claims 1 to 3 or the animal tissue embedding agent prepared by the preparation method according to any one of claims 4 to 6.

9. The method for freezing and embedding animal tissue according to claim 8, characterized in that: The animal tissues are washed with pre-cooled PBS or physiological saline before freezing and embedding.

10. The method for freezing and embedding animal tissue according to claim 9, characterized in that: The step of removing air bubbles from the embedding medium before freezing is also included.

Images