A frozen embedding method for effectively improving OCT infiltration of mouse lung tissue

Abstract

The application discloses a frozen embedding method for effectively improving OCT infiltration of mouse lung tissues, and mainly relates to the technical field of experimental animal tissue sections. The frozen embedding method for effectively improving OCT infiltration of mouse lung tissues comprises the following steps: placing mouse lungs in a frozen section embedding agent for first vacuumizing treatment before perfusion; adopting a needle to perforate the surface of a lung lobe for hole perforating treatment after perfusion; placing the mouse lungs in the frozen section embedding agent for second vacuumizing treatment; and finally immersing the mouse lungs in a frozen section perfusion liquid for frozen embedding, so as to obtain frozen embedded lung tissues, effectively improve OCT infiltration of mouse lung tissues, and solve the problems of insufficient support of the edge of a lung lobe and separation of visceral pleura.

Description

Technical Field

[0001] This invention relates to the field of laboratory animal tissue sectioning technology, and in particular to a cryo-embedding method that effectively improves OCT infiltration in mouse lung tissue. Background Technology

[0002] Frozen section preparation involves rapidly freezing fresh tissue with cryo-embedding material (OCT) at low temperatures and then slicing it into thin sections. Compared to the commonly used paraffin sectioning method, frozen sectioning is quick and simple, requires no treatment with organic reagents such as ethanol or xylene, and can better preserve enzyme activity and antigen immunoreactivity. Therefore, it is widely used in research such as immunohistochemistry, immunofluorescence, enzyme detection, tissue localization, in situ hybridization, and spatiotemporal transcriptome sequencing.

[0003] Mouse lung tissue is an important experimental material for studying respiratory diseases. Unlike solid organs such as the heart, liver, and kidneys, lung tissue contains gas-filled cavities such as bronchial lumens and alveolar cavities. During the preparation of ordinary frozen sections, insufficient support can easily lead to problems such as incomplete sections, knife marks, ice crystals, wrinkles, alveolar collapse, or tissue deformation. Existing methods have attempted to improve lung tissue morphology by perfusing OCT followed by vacuuming; however, poor OCT infiltration and visceral pleura detachment may still exist in the distal part of mouse lung tissue. Poor OCT infiltration can cause ice crystals to form in uninfiltrated areas during freezing, damaging cell membrane integrity. Visceral pleura detachment can easily cause tissue wrinkling or deformation during mounting, affecting the accuracy of subsequent spatial transcriptome sequencing, staining observation, and other experimental results. Summary of the Invention

[0004] This invention proposes a cryo-embedding method that effectively improves the OCT infiltration of mouse lung tissue, solving the technical problems of poor OCT infiltration, insufficient support at the lung lobe edge, and easy separation of the visceral pleura from the lung tissue during the preparation of frozen sections of mouse lung tissue.

[0005] To achieve the above objectives, this invention proposes a cryoemulation method for effectively improving OCT infiltration in mouse lung tissue. The cryoemulation method for effectively improving OCT infiltration in mouse lung tissue is characterized by comprising the following steps: S10. The mouse lungs were placed in the frozen section embedding medium for the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, perfuse them with frozen section embedding medium, and puncture the surface of the lung lobes with a needle. S30. Remove the mouse lungs after the puncture treatment and place them in frozen section embedding medium for a second vacuum treatment. S40. The mouse lungs that have undergone the second vacuum treatment are removed and placed in frozen section perfusion fluid for cryopreservation to obtain frozen embedded mouse lung tissue.

[0006] In one embodiment, in steps S10, S20, and S30, the frozen section embedding agent is an OCT solution with a concentration of 45-55%.

[0007] In one embodiment, step S10, the first vacuuming process includes the following steps: vacuuming for 40-80 seconds, releasing gas for 4-6 minutes, vacuuming for 80-100 seconds, and releasing gas for 4-6 minutes to complete the first vacuuming process.

[0008] In one embodiment, in step S20, the diameter of the needle is 0.1-0.5 mm.

[0009] In one embodiment, in step S20, the surface of the lung lobe is a concave surface.

[0010] In one embodiment, step S30 includes the following steps: stop vacuuming after 120-150 seconds, release air for 4-6 minutes, repeat 3-5 times until the white patches inside the lung lobe disappear, thus completing the second vacuuming process.

[0011] In one embodiment, in step S40, the frozen section perfusion solution is an OCT solution with a concentration of 45-55%.

[0012] In one embodiment, the OCT solution comprises pure OCT solution, PBS, and an RNase inhibitor.

[0013] In one embodiment, the volume ratio of the pure OCT solution, PBS, and RNase inhibitor is 8-12:7-11:1.

[0014] This invention proposes an effective frozen embedding method for improving OCT infiltration in mouse lung tissue, comprising the following steps: The entire mouse lung is harvested and placed in frozen section embedding medium for vacuum treatment. This first vacuum treatment before perfusion removes existing gas from the lung tissue and bronchi, facilitating subsequent OCT entry into the alveolar cavity and bronchial terminals via the main trachea. The lung is then removed and perfused with frozen section embedding medium via the main trachea. After the first vacuum treatment, perfusion is performed, allowing the OCT to fill the main cavity of the lung tissue and provide support for subsequent frozen sections. Several small holes are lightly punctured on the concave surface of the lung lobe edge using a needle, and the lung is placed in frozen section embedding medium again for a second vacuum treatment. These small vent holes provide an outlet for residual gas squeezed to the bronchioles after perfusion. The second vacuum treatment further promotes the expansion of residual gas and its escape from the vent holes, allowing the OCT to infiltrate the distal lung tissue and the lung lobe edge. Finally, the lungs were immersed in the frozen section perfusion solution for cryopreservation, maintaining the morphology of the lung tissue and forming a support for the section, thus obtaining frozen-embedded lung tissue. Through a first vacuuming before perfusion, puncturing and venting after perfusion, and a second extreme vacuuming, the original gas in the lung tissue and residual gas pushed to the terminal bronchioles by OCT can be fully expelled, allowing OCT to more fully infiltrate the distal lung tissue and the edge regions of the lung lobes. Using the same OCT for perfusion, vacuuming, and embedding also reduces the risk of visceral pleural detachment caused by the concentration difference of OCT inside and outside the lung, thereby obtaining frozen sections of mouse lung tissue with more intact morphology and better RNA protection. This solves the problems of poor OCT infiltration, insufficient support of the lung lobe edges, and visceral pleural detachment in existing frozen sections of mouse lung tissue, reducing problems such as airway tearing, alveolar collapse, and tissue deformation. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 A schematic diagram illustrating poor OCT infiltration and detachment of the visceral pleura; Figure 2 This is a schematic diagram showing the HE staining results of frozen-embedded lung tissue sections prepared for Comparative Example 1. Figure 3 This is a schematic diagram showing the HE staining results of frozen-embedded lung tissue sections prepared in Comparative Example 2. Figure 4This is a schematic diagram showing the HE staining results of frozen-embedded lung tissue sections prepared in Comparative Example 3. Figure 5 This is a schematic diagram showing the HE staining results of frozen-embedded lung tissue sections prepared in Example 2. Figure 6 This is a schematic diagram showing the HE staining results of frozen-embedded lung tissue sections prepared in Example 1. Figure 7 This is a schematic diagram of the RNA quality control results for the sample corresponding to Example 1; Figure 8 This is a schematic diagram of the RNA quality control results for the sample corresponding to Example 3; Figure 9 This is a schematic diagram showing the comparison of RQS values ​​between the sample corresponding to Example 1 and the sample corresponding to Example 3.

[0017] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Mouse lung tissue is a commonly used experimental material in respiratory disease research. However, unlike solid organs such as the heart, liver, and kidneys, lung tissue contains numerous gas-filled cavities, including bronchial lumens and alveolar cavities. Conventional frozen section embedding methods often fail to allow OCT to fully penetrate the distal lung tissue and the edge regions of the lung lobes, easily leading to problems such as poor OCT infiltration, ice crystal damage, insufficient support during sectioning, and visceral pleural detachment. Visceral pleural detachment refers to the phenomenon of separation and splitting at the interface between the visceral pleura and the lung during frozen section preparation. Figure 1As shown, this phenomenon easily leads to tissue wrinkling or deformation during mounting, damaging the original structure of the tissue and affecting the accuracy of subsequent morphological observation and spatial transcriptome sequencing results. Simultaneously, poor OCT infiltration leaves cells in un-OCT-infiltrated areas without the protection of RNase inhibitors, increasing the risk of RNA degradation and potentially causing ice crystal formation during subsequent freezing, damaging the integrity of cell and nuclear membranes, releasing RNA extracellularly, and causing RNA contamination between adjacent cells, further affecting the accuracy of spatial transcriptome sequencing results. Therefore, there is an urgent need for an embedding method that can promote the expulsion of residual gas from lung tissue, improve OCT infiltration, reduce visceral pleural detachment, and maintain the structural integrity of frozen lung tissue sections.

[0020] In view of this, the present invention proposes a method for cryopreservation that effectively improves OCT infiltration in mouse lung tissue, comprising the following steps: S10. The mouse lungs were placed in the frozen section embedding medium for the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, perfuse them with frozen section embedding medium, and puncture the surface of the lung lobes with a needle. S30. Remove the mouse lungs after the puncture treatment and place them in frozen section embedding medium for a second vacuum treatment. S40. The mouse lungs that have undergone the second vacuum treatment are removed and placed in frozen section perfusion fluid for cryopreservation to obtain frozen embedded mouse lung tissue.

[0021] This invention obtains frozen-embedded mouse lung tissue through a process involving a first vacuum treatment before perfusion, venting after perfusion, a second extreme vacuum treatment, and embedding and perfusion with OCT at the same concentration. In step S10, the first vacuum treatment before perfusion removes existing gas from the lung tissue and bronchi, facilitating the subsequent entry of OCT into the alveolar cavity and bronchial terminals via the main trachea. In step S20, perfusion is performed after the vacuum treatment. After perfusion of the frozen section embedding agent through the main trachea, the OCT fills the main cavity of the lung tissue and provides support for subsequent frozen sections. Furthermore, venting is performed on the surface of the lung lobes before the second vacuum treatment, creating tiny vent holes on the concave surface of the lobe edges to allow residual gas squeezed to the bronchioles after perfusion to escape. After the initial vacuuming process, very little residual gas remains inside the lungs. However, after OCT perfusion, some of this residual gas is compressed to the ends of the bronchioles. This residual gas hinders OCT infiltration to the lung margins. Therefore, a second vacuuming process (S30) is implemented to further promote the expansion of the residual gas and its escape from the vent, allowing OCT to infiltrate the distal lung tissue and the edges of the lung lobes. In step S40, OCT of the same concentration is used for embedding, eliminating the concentration difference of OCT inside and outside the lungs and resolving the issues of poor OCT infiltration and visceral pleural detachment in frozen sections of mouse lung tissue. This ultimately solves the problem of obtaining intact frozen sections of lung tissue due to the lack of sufficient support during slicing, as lung tissue is not a solid tissue and contains gas-filled cavities such as bronchial lumens and alveolar cavities.

[0022] In the following embodiments, in steps S10, S20, and S30, the frozen section embedding agent is an OCT solution with a concentration of 45-55%.

[0023] Using a 45-55% concentration of OCT solution as the embedding medium for frozen sections achieves a balance between perfusion fluidity and embedding support. This concentration range avoids the problem of pure OCT having excessive viscosity, making it difficult to fully perfuse to the distal lung tissue via the main trachea, while also providing good structural support for the alveolar cavities, bronchial cavities, and lobar edges after freezing, which is beneficial for obtaining frozen sections of lung tissue with intact morphology and well-infiltrated margins.

[0024] In the following embodiment, step S10 includes the following steps: vacuuming for 40-80 seconds, releasing gas for 4-6 minutes, vacuuming for 80-100 seconds, and releasing gas for 4-6 minutes to complete the first vacuuming process.

[0025] The initial vacuuming process employs alternating vacuuming and venting, pre-emptively removing easily expelled gases from the lung tissue before OCT perfusion, making the internal cavities of the lung tissue easier for OCT to penetrate. This facilitates the gradual removal of residual gases from the lung tissue without excessively damaging its structure. Segmented vacuuming and venting ensure relatively gentle pressure changes inside and outside the lung tissue, making it easier for the lung tissue to be filled with the embedding agent during subsequent OCT perfusion.

[0026] In the following embodiments, in step S20, the diameter of the needle is 0.1-0.5 mm.

[0027] The needle diameter is controlled between 0.1-0.5 mm, forming tiny vent holes on the surface of the lung lobe for residual gas to escape. This small diameter range will not cause significant mechanical damage to the overall structure of the lung lobe, while reducing the risk of lung tissue tearing or local structural damage caused by excessively large vent holes. It can balance the venting effect and tissue integrity.

[0028] In the following embodiment, in step S20, the surface of the lung lobe is a concave surface.

[0029] Choosing the puncture site on the concave surface of the lung lobe helps to create an exhaust channel without significantly damaging the shape of the lung lobe and the main observation area; it also makes it easier for residual gas to be discharged from the edge and end of the lung lobe during the second vacuuming process, avoiding alveolar rupture or cavitation caused by local gas expansion during the extreme vacuuming process of the second vacuuming, thereby maintaining the high infiltration effect of OCT at the edge of the lung lobe and the integrity of the tissue structure.

[0030] In the following embodiment, step S30 includes the following steps: stop vacuuming after 120-150 seconds, release air for 4-6 minutes, repeat 3-5 times until the white patches inside the lung lobe disappear, thus completing the second vacuuming process.

[0031] At this point, very little residual gas remains inside the lungs. A very low vacuum is required for this remaining gas to expand and escape. However, if the vacuum is too low, the OCT (occlusive tissue membrane) inside the lungs will boil, generating a large amount of gas that could cause alveolar rupture. Therefore, the limit for vacuuming is to stop when the OCT is about to boil. Thus, the second vacuuming process uses the near-boiling point of the OCT as the criterion for stopping vacuuming, combined with venting for 4-6 minutes and 3-5 cycles. This achieves a lower vacuum to expel residual gas from the ends of the bronchioles while preventing the OCT from continuously boiling and generating a large amount of gas that could lead to alveolar rupture or cavitation. This treatment helps to further alleviate the problem of poor OCT infiltration at the lung lobe edges.

[0032] The reason for setting up a second vacuuming process is that after the first vacuuming and main tracheal perfusion, a small amount of residual gas may still remain inside the lungs. This gas is easily pushed and trapped at the ends of the bronchioles by the perfused OCT, directly affecting the OCT's continued infiltration into the lung lobe edges and distal lung tissue. The second vacuuming process reduces the vacuum level to the critical state where the OCT is about to boil by vacuuming for 120-150 seconds, allowing the small amount of residual gas to fully expand and be discharged through the venting channels formed by the puncture. At the same time, stopping the vacuuming and releasing the gas when the OCT is about to boil can prevent the OCT from continuously boiling and generating a large number of bubbles, which could cause alveolar rupture or cavitation. Therefore, this step can further improve OCT infiltration while protecting the lung tissue structure, and reduce the problem of poor OCT infiltration at the lung lobe edges and visceral pleural detachment.

[0033] In the following embodiments, in step S40, the frozen section perfusion fluid is an OCT solution with a concentration of 45-55%.

[0034] In step S40, a frozen section perfusion medium with a concentration of 45-55% OCT solution is used, and the OCT concentration is consistent with that of the frozen section embedding medium in S10, S20, and S30. This eliminates the concentration difference of OCT inside and outside the lung and reduces the risk of the lung section edge detaching from the OCT or visceral pleural tuberculosis interface due to the concentration difference.

[0035] In the following examples, the OCT solution comprises pure OCT solution, PBS, and RNase inhibitor.

[0036] The OCT solution was obtained by diluting pure OCT with PBS, where PBS was used to dilute OCT to obtain a fluidity suitable for perfusion, and pure OCT was used to provide support properties after cryopreservation. At the same time, in order to prevent RNA degradation in the lungs, RNase inhibitors were added to reduce the risk of sample RNA degradation, which is beneficial to maintaining the RNA integrity in subsequent nucleic acid detection experiments such as spatial transcriptome sequencing.

[0037] In the following examples, the volume ratio of the pure OCT solution, PBS, and RNase inhibitor is 8-12:7-11:1.

[0038] The volume ratio of pure OCT solution, PBS, and RNase inhibitor was controlled at 8-12:7-11:1, which ensured that the OCT solution was maintained within a concentration range suitable for perfusion, vacuum infiltration, and cryopreservation, while also providing support after freezing and protecting the integrity of RNA in lung tissue.

[0039] Experimental materials The preparation method of 50% OCT containing RNase inhibitor is as follows: Take pure OCT solution, PBS and RNase inhibitor, and mix them evenly in a volume ratio of 10:9:1 to obtain the 50% OCT containing RNase inhibitor; wherein, pure OCT solution accounts for 50% of the total volume of the mixture.

[0040] The preparation method of 20% OCT is as follows: Take pure OCT solution, PBS and RNase inhibitor, and mix them evenly in a volume ratio of 2:7:1 to obtain the 20% OCT; wherein, pure OCT solution accounts for 20% of the total volume of the mixture.

[0041] The preparation method of 50% OCT without RNase inhibitors is as follows: Take pure OCT solution and PBS, and mix them evenly at a volume ratio of 1:1 to obtain the 50% OCT without RNase inhibitor; wherein, pure OCT solution accounts for 50% of the total volume of the mixture.

[0042] The steps for removing mouse lungs are as follows: C57 mice were euthanized at 3, 7, 14, and 28 days of age, and the entire lungs, including the main trachea, were completely removed. The removed lungs were then placed in nuclease-free petri dishes for later use, thus obtaining the mouse lungs. During the procedure, the main trachea structure was kept intact so that frozen section embedding medium could be perfused through the main trachea.

[0043] The steps for removing the lungs from tracheotomized mice are as follows: (1) After euthanizing C57 mice at 3, 7, 14 and 28 days of age, the thyroid gland and esophagus were removed with wide-tipped forceps and surgical scissors to fully expose the main trachea and the thoracic cavity was opened. (2) After inserting the syringe needle into the main trachea, the tracheal wall was tied to the syringe needle with surgical suture. Then, 20% OCT was perfused into the lungs according to the age of the mice to fully fill the lungs. The OCT perfusion volumes for mice at 3 days, 7 days, 14 days and 28 days were 0.08 mL, 0.16 mL, 0.4 mL and 0.8 mL, respectively. (3) After the entire lung is filled with OCT, the needle is removed, and the trachea is tied tightly below the tracheal puncture to prevent OCT leakage. The entire lung is then removed and placed in a nuclease-free petri dish. The left lung lobe is carefully removed to obtain the lung of the tracheotomy mouse. The difference between the lung of the tracheotomy mouse and the lung of a mouse is that: before removing the lung of the tracheotomy mouse, 20% OCT is perfused into the lung.

[0044] Example 1 A cryo-embedding method for effectively improving OCT infiltration in mouse lung tissue includes the following steps: S10. The mouse lungs were placed in 50% OCT frozen section embedding medium containing RNase inhibitors for the first vacuum treatment. The first vacuum treatment method was to vacuum for 60s, release for 5min, vacuum for 90s, and release for 5min to complete the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, infuse 50% OCT frozen section embedding medium containing RNase inhibitors through the main trachea, and puncture the concave surface of the lung lobe with a needle with a diameter of 0.3 mm. S30. Remove the mouse lungs after the puncture treatment and place them in 50% OCT frozen section embedding medium containing RNase inhibitor for a second vacuum treatment. The second vacuum treatment method is to stop vacuuming after 135 seconds, release the gas for 5 minutes, and repeat 4 times until the white patches inside the lung lobe disappear under a vacuum of 6 hPa, thus completing the second vacuum treatment. S40. The mouse lungs that have undergone the second vacuum treatment are removed and placed in 50% OCT frozen section perfusion solution containing RNase inhibitors for cryo-embedding. Before cryo-embedding, the steps include adjusting the tissue position, removing air bubbles, and fusing the tissue and 50% OCT frozen section perfusion solution containing RNase inhibitors on ice for 5 minutes. The cryo-embedding process is carried out on dry ice to obtain cryo-embedded mouse lung tissue.

[0045] Comparative Example 1 A method for cryo-embedding includes the following steps: S10. The lungs of tracheotomized mice were placed in 20% OCT frozen section embedding medium and vacuumed. The vacuuming method was to vacuum for 60s, release for 5min, vacuum for 90s, and release for 5min to complete the vacuuming process. S20. The mouse lungs that have undergone vacuum treatment are removed and placed in 100% OCT frozen section perfusion solution for cryo-embedding. Before cryo-embedding, the steps include adjusting the tissue position, removing air bubbles, and allowing the tissue and 100% OCT frozen section perfusion solution to fuse on ice for 5 minutes. The cryo-embedding process is carried out on dry ice to obtain cryo-embedded mouse lung tissue.

[0046] Comparative Example 2 A method for cryo-embedding includes the following steps: S10. The mouse lungs were placed in 20% OCT frozen section embedding medium for the first vacuum treatment. The first vacuum treatment method was to vacuum for 60s, release for 5min, vacuum for 90s, and release for 5min to complete the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment and infuse them with 20% OCT frozen section embedding medium through the main trachea to fill the lungs. S30. Remove the perfused mouse lungs and place them in 100% OCT frozen section perfusion solution for cryo-embedding. Before cryo-embedding, the procedure includes adjusting the tissue position, removing air bubbles, and allowing the tissue and 100% OCT frozen section perfusion solution to fuse on ice for 5 minutes. The cryo-embedding process is performed entirely on dry ice to obtain cryo-embedded mouse lung tissue.

[0047] Comparative Example 3 A method for cryo-embedding includes the following steps: S10. The mouse lungs were placed in 20% OCT frozen section embedding medium for the first vacuum treatment. The first vacuum treatment method was to vacuum for 60s, release for 5min, vacuum for 90s, and release for 5min to complete the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, and infuse them with 20% OCT frozen section embedding medium through the main trachea to fill the lungs. Then, place the perfused lungs in 20% OCT frozen section embedding medium for a second vacuum treatment. The second vacuum treatment method is to stop vacuuming after 135 seconds, release the gas for 5 minutes, and repeat the cycle 4 times until the white patches inside the lung lobe disappear under a vacuum of 6 hPa, thus completing the second vacuum treatment. S30. The mouse lungs that have undergone the second vacuum treatment are removed and placed in 100% OCT frozen section perfusion solution for cryo-embedding. Before cryo-embedding, the steps include adjusting the tissue position, removing air bubbles, and allowing the tissue and 100% OCT frozen section perfusion solution to fuse on ice for 5 minutes. The cryo-embedding process is carried out on dry ice to obtain cryo-embedded mouse lung tissue.

[0048] Example 2 A method for cryo-embedding includes the following steps: S10. The mouse lungs were placed in 20% OCT frozen section embedding medium for the first vacuum treatment. The first vacuum treatment method was to vacuum for 60s, release for 5min, vacuum for 90s, and release for 5min to complete the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, infuse 20% OCT frozen section embedding medium through the main trachea, and puncture the concave surface of the lung lobe with a needle with a diameter of 0.3 mm. S30. Remove the mouse lungs after the puncture treatment and place them in 20% OCT frozen section embedding medium for a second vacuum treatment. The second vacuum treatment method is to stop vacuuming after 135 seconds, release air for 5 minutes, and repeat 4 times until the white patches inside the lung lobe disappear under a vacuum of 6 hPa, thus completing the second vacuum treatment. S40. The mouse lungs that have undergone the second vacuum treatment are removed and placed in 100% OCT frozen section perfusion solution for cryo-embedding. Before cryo-embedding, the steps include adjusting the tissue position, removing air bubbles, and allowing the tissue and 100% OCT frozen section perfusion solution to fuse on ice for 5 minutes. The cryo-embedding process is carried out on dry ice to obtain cryo-embedded mouse lung tissue.

[0049] Example 3 A method for cryo-embedding includes the following steps: S10. The mouse lungs were placed in 50% OCT frozen section embedding medium without RNase inhibitors for the first vacuum treatment. The first vacuum treatment method was to vacuum for 60s, release for 5min, vacuum for 90s, and release for 5min to complete the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, infuse 50% OCT frozen section embedding medium without RNase inhibitors through the main trachea, and puncture the concave surface of the lung lobe with a needle with a diameter of 0.3 mm. S30. Remove the mouse lungs after the puncture treatment and place them in 50% OCT frozen section embedding medium without RNase inhibitors for a second vacuum treatment. The second vacuum treatment method is to stop vacuuming after 135 seconds, release the gas for 5 minutes, and repeat 4 times until the white patches inside the lung lobe disappear under a vacuum of 6 hPa, thus completing the second vacuum treatment. S40. The mouse lungs that have undergone the second vacuum treatment are removed and placed in 50% OCT frozen section perfusion fluid without RNase inhibitors for cryo-embedding. Before cryo-embedding, the steps include adjusting the tissue position, removing air bubbles, and fusing the tissue and 50% OCT frozen section perfusion fluid without RNase inhibitors on ice for 5 minutes. The cryo-embedding process is carried out on dry ice to obtain cryo-embedded mouse lung tissue.

[0050] The difference between Comparative Example 1 and Comparative Example 2 is that: in Comparative Example 1, the lungs of mice without trachea were first perfused with 20% OCT and then subjected to the first vacuum treatment; in Comparative Example 2, the lungs of mice were first subjected to the first vacuum treatment and then perfused with 20% OCT, while the other experimental conditions were the same. The difference between Comparative Example 2 and Comparative Example 3 is that Comparative Example 3 adds a second vacuuming step after infusion, while Comparative Example 2 only completes the first vacuuming and infusion, without the second vacuuming operation, and the other experimental conditions are the same. The difference between Comparative Example 3 and Example 2 is that Example 2 added a perforation treatment on the concave surface of the lung lobe before the second vacuuming, while Comparative Example 3 did not perform the perforation treatment, and the other experimental conditions were the same. The difference between Example 1 and Example 2 is as follows: In Example 1, both the OCT frozen section embedding medium and the OCT frozen section perfusion medium used 50% OCT containing RNase inhibitors, and the same concentration of OCT solution was used throughout the process; In Example 2, the OCT frozen section embedding medium and the OCT frozen section perfusion medium used 20% OTC and 100% OCT, respectively, and the other experimental conditions were the same. The difference between Examples 1 and 3 is that the 50% OCT in Example 1 contains an RNase inhibitor, and the volume ratio of pure OCT:PBS:RNase inhibitor in its formula is 10:9:1; the 50% OCT in Example 3 does not contain an RNase inhibitor, and the volume ratio of pure OCT:PBS in its formula is 10:10. The other experimental conditions are the same. The specific differences between the examples and the comparative examples are shown in Table 1.

[0051] Table 1. Differences between the Examples and Comparative Examples

[0052] Test Example 1 The frozen-embedded mouse lung tissues obtained in Comparative Examples 1-2 were sectioned and subjected to HE staining tests to observe the effects of different vacuuming times on the OCT infiltration effect and section morphology of the lung tissues.

[0053] The results show that: Figure 2 and Figure 3 As shown, compared with Comparative Example 1, which involved vacuuming after OCT perfusion, Comparative Example 2, where vacuuming was performed before OCT perfusion, showed an overall reduction in poor OCT infiltration at the lung lobe edges. Significant improvement was observed in the lung tissue of mice at 14 and 28 days, with some improvement also observed at 3 and 7 days. This indicates that vacuuming before perfusion facilitates the expulsion of gas from the terminal bronchioles without OCT obstruction.

[0054] Test Example 2 The frozen-embedded mouse lung tissues obtained in Comparative Examples 2-3 were sectioned and subjected to HE staining tests to observe the effect of adding a second vacuum treatment on the OCT infiltration effect of lung tissue.

[0055] The results show that: Figure 3 and Figure 4 As shown, compared with Comparative Example 2, which only underwent the first vacuum treatment before perfusion, Comparative Example 3, after adding the second vacuum treatment, showed that the poor OCT infiltration at the edge of the mouse lung tissue sections at 14 and 28 days had basically disappeared, and the poor OCT infiltration at the edge of the mouse lung tissue sections at 3 and 7 days had also been significantly reduced. This indicates that the second vacuum treatment can promote the further expulsion of gas remaining at the end of the bronchioles after perfusion.

[0056] Test Example 3 The frozen-embedded mouse lung tissues obtained in Comparative Example 3 and Example 2 were sectioned and subjected to HE staining tests to observe the effect of adding concave puncture treatment on lung tissue structure integrity and OCT infiltration effect before the second vacuuming.

[0057] The results show that: Figure 4 and Figure 5 As shown, compared with Comparative Example 3 which did not undergo puncture treatment, after forming an exhaust hole on the concave surface of the lung lobe in Example 2, the local cavitation phenomenon caused by extreme vacuuming disappeared, and the poor OCT infiltration of the lung lobe edge was further reduced. This indicates that puncture treatment can provide an exhaust channel for residual gas and reduce the risk of alveolar rupture caused by local gas expansion.

[0058] Test Example 4 The frozen-embedded mouse lung tissues obtained in Examples 1-2 were sectioned and subjected to HE staining tests to observe the effects of perfusion, vacuuming, and OCT concentration adjustment on visceral pleural detachment and lung lobe edge infiltration.

[0059] The results show that: Figure 5 and Figure 6 As shown, compared with Example 2, which used 20% OCT perfusion, vacuuming, and 100% OCT embedding, Example 1, which used 50% OCT containing RNase inhibitors for perfusion, vacuuming, and embedding, showed that the detachment of the lung slice edges from the OCT and the detachment of the visceral pleura basically disappeared. This indicates that using an OCT system with the same or similar concentration can reduce the concentration difference of OCT inside and outside the lung and improve the integrity of the slice morphology.

[0060] Test Example 5 RNA extraction and quality control tests were performed on the frozen-embedded mouse lung tissues obtained in Examples 1 and 3 to evaluate the effect of adding RNase inhibitors to the OCT solution on the integrity of the sample RNA.

[0061] The results show that: Figure 7-8 The comparison shows that Example 1, which contains an RNase inhibitor, is superior. Figure 7 The RNA integrity of the corresponding sample was superior to that of Example 3, which did not contain an RNase inhibitor. Figure 8 ),pass Figure 9 The comparison shows that the RQS value of Example 1 is significantly increased, indicating that adding RNase inhibitors to the OCT solution in Example 1 can reduce the risk of RNA degradation during lung tissue processing and cryopreservation, which is beneficial for subsequent nucleic acid detection experiments such as spatial transcriptome sequencing.

[0062] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for cryo-embedding effectively improving OCT infiltration in mouse lung tissue, characterized in that, Includes the following steps: S10. The mouse lungs were placed in the frozen section embedding medium for the first vacuum treatment. S20. Remove the mouse lungs after the first vacuum treatment, perfuse them with frozen section embedding medium, and puncture the surface of the lung lobes with a needle. S30. Remove the mouse lungs after the puncture treatment and place them in frozen section embedding medium for a second vacuum treatment. S40. The mouse lungs that have undergone the second vacuum treatment are removed and placed in frozen section perfusion fluid for cryopreservation to obtain frozen embedded mouse lung tissue.

2. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 1, characterized in that, In steps S10, S20, or S30, the frozen section embedding agent is an OCT solution with a concentration of 45-55%.

3. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 1, characterized in that, In step S10, the first vacuuming process includes the following steps: vacuuming for 40-80 seconds, releasing gas for 4-6 minutes, vacuuming for 80-100 seconds, and releasing gas for 4-6 minutes to complete the first vacuuming process.

4. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 1, characterized in that, In step S20, the diameter of the needle is 0.1-0.5 mm.

5. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 1, characterized in that, In step S20, the surface of the lung lobe is a concave surface.

6. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 1, characterized in that, In step S30, the second vacuuming process The procedure includes the following steps: stop vacuuming after 120-150 seconds, release air for 4-6 minutes, repeat 3-5 times until the white patches inside the lungs disappear, and the second vacuuming process is complete.

7. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 1, characterized in that, In step S40, the frozen section perfusion solution is an OCT solution with a concentration of 45-55%.

8. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 2 or 7, characterized in that, The OCT solution includes pure OCT solution, PBS, and RNase inhibitor.

9. The cryopreservation method for effectively improving OCT infiltration in mouse lung tissue as described in claim 8, characterized in that, The volume ratio of the pure OCT solution, PBS, and RNase inhibitor is 8-12:7-11:1.

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