Embedding mold, embedding method, and related applications

Abstract

The application provides an embedding mold, an embedding method and related applications. The embedding mold is in a square funnel shape with a closed bottom surface, and a scale line is marked on the inner wall of the embedding mold. By setting the mold in a square funnel shape with a closed bottom surface, the opening gradually increases from the bottom to the top, so that the embedding of tissues of different sizes can be easily met, and the application range or use flexibility of the mold is improved. By marking a scale line on the inner wall of the mold, embedding agent can be laid at a specific scale position in advance according to the size of the tissue to be embedded when embedding in the mold, which is also conducive to keeping the tissue material in the obtained tissue embedding block in a balanced state rather than an inclined state, and then conducive to obtaining complete sections or sections of specific planes.

Description

Technical Field

[0001] This invention relates to the field of embedding sectioning technology, and more specifically, to an embedding mold, an embedding method, and related applications. Background Technology

[0002] Embedding and sectioning techniques have been widely used in various clinical pathological tissue staining processes, playing a vital role in disease diagnosis and treatment. Embedding and sectioning techniques include paraffin-embedded sections and cryo-embedded sections. Compared to paraffin-embedded sections, cryo-embedded sections offer advantages such as simpler and faster preparation, immunity to the high temperatures of paraffin infiltration and embedding, and better preservation of tissue bioactivity. This has led to their further application in basic life science research, such as using spatial transcriptome sequencing to elucidate the fundamental mechanisms of life regulatory networks in heterogeneous tissues.

[0003] Cryoemulation and sectioning is a crucial experimental technique in this emerging research method, closely related to the success of subsequent experiments and the interpretation of sequencing data. Therefore, optimizing embedding tools, embedding conditions, and subsequent sectioning operations based on existing cryoemulation techniques is essential for promoting its wider application.

[0004] Currently, commonly used cryo-embedding and sectioning methods primarily involve rapid freezing of samples using liquid nitrogen combined with isopentane. There are three commonly used embedding tools, all with fixed dimensions:

[0005] 1) Commercially available specialized embedding cassettes typically range in length and width from 5mm to 20mm and in depth from 5mm to 12mm. Due to their shallow depth, they are suitable for embedding smaller tissues, but they cannot completely cover larger tissues (thickness exceeding 1cm and area exceeding 2cm²), thus limiting their further applications.

[0006] 2) Due to the above limitations, some researchers have attempted to embed the OCT using cell culture dishes, typically 35 cm in size. This allows for complete coverage of even large tissue volumes. However, during subsequent sectioning, excess OCT around the tissue needs to be trimmed. Since the OCT solidifies into a milky white layer covering the tissue, its location cannot be directly observed. There is a risk of accidentally cutting into the tissue during trimming. This method is relatively cumbersome and may lead to experimental failure.

[0007] 3) Some researchers also use aluminum foil to make their own embedding boxes, which can be made in different sizes as needed. However, aluminum foil is not rigid enough and is easily deformed, which is not conducive to subsequent operations.

[0008] The following are examples of existing cryo-embedding and sectioning procedures:

[0009] Reagents and consumables: embedding medium OCT, isopentane, liquid nitrogen, embedding cassette (purchase commercial embedding cassette, cell culture dish or make your own aluminum foil box).

[0010] Instruments and tools: fume hood, 150ml large beaker, 40ml small beaker, tweezers, marker, small cutting knife.

[0011] 1) Pre-cool the embedding medium OCT on ice for 10 minutes. Except for sectioning, all the following operations are performed in a fume hood.

[0012] 2) Prepare two stainless steel beakers, one large and one small (150ml for the large beaker and 40ml for the small beaker). Add 1 / 3 liquid nitrogen to the large beaker and 1 / 3 isopentane to the small beaker. Place the small beaker containing isopentane into the large beaker containing liquid nitrogen and incubate for at least 10 minutes. Pre-cool the isopentane.

[0013] 3) Select a tissue embedding cassette based on the size of the embedded tissue (purchase a dedicated embedding cassette, cell culture dish, or make your own foil cassette).

[0014] 4) Add a layer of OCT about 3 mm thick to the embedding cassette and freeze it in isopentane until the OCT hardens, keeping the OCT surface as flat as possible.

[0015] 5) Take a tissue sample, pre-determine the tissue embedding direction, and gently pick up the tissue with tweezers and place it on the pre-laid OCT in the embedding cassette.

[0016] 6) Adjust the direction and add pre-cooled OCT to completely cover the tissue. Avoid generating air bubbles during this process, especially near the tissue.

[0017] 7) Use tweezers to pick up the embedding box and place it in isopentane that has been pre-cooled with liquid nitrogen, and wait for the OCT to harden completely.

[0018] 8) Once tissue embedding is complete, mark the tissue orientation on all four sides of the embedding cassette and place it at -80 degrees, or proceed directly to sectioning.

[0019] 9) Place the embedded tissue in a cryostat for equilibration for 30 minutes.

[0020] 10) Use tweezers to insert into the OCT containing the tissue sample and remove it from the embedding cassette, being careful not to insert it into the tissue sample.

[0021] 11) Use a knife to cut and modify the OCT around the embedded tissue to facilitate subsequent sectioning. Be careful not to cut the sample.

[0022] 12) Place the modified OCT block containing the tissue onto a frozen sample holder covered with a layer of liquid OCT, and then perform rapid freezing fixation.

[0023] 13) After the OCT of the tissue sample is completely fixed, fix the tissue block onto the frozen head and section it.

[0024] 14) After the slides are mounted onto glass slides (hereinafter referred to as slides), they are subjected to hematoxylin-eosin staining (HE staining) for detection, or mounted onto spatiotemporal microarrays for subsequent spatiotemporal sequencing.

[0025] The above method has the following disadvantages: (1) The tissue is frozen using liquid nitrogen combined with isopentane. Since isopentane has certain toxicity, it needs to be carried out in a fume hood, which is not friendly to laboratory operations without fume hoods and the operation is relatively cumbersome; (2) Existing embedding tools all have certain disadvantages.

[0026] a. Purchasing specialized embedding cassettes from the market – these have fixed dimensions and are typically shallow (5mm-12mm), unsuitable for embedding larger tissue samples. A common problem is that after adding an OCT layer to the bottom of the mold, the tissue thickness exceeds the mold's depth, making it impossible to completely cover the tissue with additional OCT. Some tissues may even exceed the width of the mold itself. Furthermore, they are difficult to remove from the embedding cassette, requiring the outer OCT layer to slightly melt before prying them out with tweezers.

[0027] b. Cell Culture Dish Embedding – The size is fixed, allowing for the embedding of larger tissues, but the diameter of the culture dishes typically used exceeds 35mm. Subsequent sectioning requires trimming away excess OCT around the tissue, which is cumbersome and time-consuming, potentially affecting experimental results. Furthermore, because the solidified OCT is milky white and covers the tissue, its location cannot be directly observed; accidental cutting during trimming can also lead to experimental failure. It is also difficult to remove; the outer layer of OCT needs to melt slightly before it can be pried out with forceps.

[0028] c. Homemade foil boxes – embedding boxes of different sizes can be made as needed, but because the material is thin, it is easy to deform and the embedding surface is uneven, which is not conducive to subsequent operations.

[0029] In addition, all three embedding tools mentioned above have some common drawbacks, such as: (1) If the contact surface with the bottom of the mold is not flat during embedding, the mold is prone to tilting, which will cause the sample to be placed at an angle, resulting in the obtained section not being the desired section (e.g. Figure 1 (1) The petri dish on the left. (2) The mold groove is not directional. Once the embedded tissue block is taken out of the mold, it loses its direction, which is not conducive to the analysis of the results later. (3) One size of embedding box is only suitable for embedding tissue of the corresponding size. When embedding tissue of different sizes, it is necessary to change to a different size of large tissue, which is not flexible enough.

[0030] Therefore, improvements are still needed to the existing cryosection embedding methods. Summary of the Invention

[0031] The main objective of this invention is to provide an embedding mold, an embedding method, and related applications to solve the problem that existing embedding molds are unable to meet the embedding requirements of tissue samples of various sizes.

[0032] To achieve the above objectives, according to one aspect of the present invention, an embedding mold is provided, which is a square funnel shape with a closed bottom, and the inner wall of the embedding mold is marked with scale lines.

[0033] Furthermore, the embedding mold includes a square base and four trapezoidal sides connected to each side of the square base. The four trapezoidal sides are connected in sequence to form a square funnel shape, and scale lines are marked on the inner wall of any one of the trapezoidal sides.

[0034] Furthermore, the area of ​​the square base is (0.5~3)cm×(0.5~3)cm.

[0035] Furthermore, the scale lines are multiple straight lines set on the inner wall of the trapezoidal side and parallel to the connected sides of the trapezoidal side, with the lengths of the multiple straight lines being 1.0cm, 1.5cm, 2cm, 2.5cm and 3cm respectively; preferably, the square base is a square, and the area of ​​the plane where the scale lines are located is 1.0cm×1.0cm, 1.5cm×1.5cm, 2cm×2cm, 2.5cm×2.5cm and 3cm×3cm respectively.

[0036] Furthermore, the embedding mold also includes a level, which is disposed on the outer wall of any one or more of the four trapezoidal sides; preferably, the level is disposed on the outer wall of the trapezoidal side using adhesive tape.

[0037] Furthermore, any one or more of the four trapezoidal sides are detachably connected by snap-fits, preferably with the snap-fits located at the top of the side where each trapezoidal side connects to the adjacent trapezoidal side.

[0038] Furthermore, the embedding mold also has an embedding direction marking angle, which is formed by a plane that covers the interior angle formed by any two adjacent trapezoidal sides, and the plane is connected to the square base.

[0039] Furthermore, the mold material is selected from PVC, PC, PE or PP.

[0040] According to a second aspect of this application, an embedding kit is provided, the kit including an embedding mold, which is any of the embedding molds described above.

[0041] Application of any of the above-mentioned embedding molds in section embedding.

[0042] Furthermore, it is used for embedding frozen sections.

[0043] According to a third aspect of this application, a cryo-embedding method is provided, the embedding method comprising embedding using any of the above-described embedding molds.

[0044] Furthermore, the embedding method includes: laying a bottom layer of OCT at the bottom of the mold up to the graduation line corresponding to the length of the tissue to be embedded; transferring the mold containing the bottom layer of OCT to crushed dry ice; placing the tissue to be embedded in the mold containing the bottom layer of OCT; covering the tissue to be embedded with an upper layer of OCT; and removing the tissue embedding block after the upper layer of OCT has solidified.

[0045] Furthermore, when transferring the mold containing the underlying OCT onto crushed dry ice, the mold is kept balanced using a level on the mold; preferably, the tissue embedding block is removed by opening the latch on the mold; preferably, the tissue embedding block has directional markings on it.

[0046] According to a fourth aspect of this application, a method for cryosectioning tissue is provided, the method comprising placing cryoembedded tissue on a cryostat for slicing, wherein the cryoembedded tissue is obtained by embedding using any of the above-described embedding methods.

[0047] Furthermore, after adjusting the frozen tissue embedding block to the predetermined direction according to the marked angle, it is fixed to the freezing head of the cryostat for sectioning, resulting in directional sections.

[0048] By applying the technical solution of this invention, this application sets the mold in the shape of a square funnel with a closed bottom, and the opening gradually increases from bottom to top, thus facilitating the embedding of tissues of different sizes and improving the applicability and flexibility of the mold. Marking graduation lines on the inner wall of the mold allows for the pre-laying of embedding agent at specific graduation positions according to the size of the tissue to be embedded during embedding. This also helps to keep the tissue material in the obtained tissue embedding block balanced rather than tilted, which is beneficial for obtaining complete sections or sections with a specific plane. Attached Figure Description

[0049] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0050] Figure 1 The illustration shows a comparison of the state of tissue material embedded in a petri dish and in a tissue block embedded using the embedding mold of this application. The tissue on the left is tilted, while the tissue on the right remains relatively horizontal and untilted.

[0051] Figure 2A schematic diagram of the embedding mold in a preferred embodiment of this application is shown.

[0052] Figure 3 The slides and HE staining results under different transport and embedding conditions are shown.

[0053] Figure 4 The slices obtained under different temperature conditions are shown.

[0054] The above figures include the following reference numerals:

[0055] 11. Scale lines; 12. Level; 13. Buckle; 14. Marking angle. Detailed Implementation

[0056] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0057] OCT embedding agent, short for optimal cutting temperature compound, is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol. It is widely used in immunohistochemistry laboratories to support tissues during frozen sectioning, increasing tissue continuity and reducing wrinkling and fragmentation. Because the OCT mixture is water-soluble, it dissolves in water during slide preparation, thus not increasing background staining during subsequent staining. Pre-impregnating tissues with the OCT mixture (optimal cutting temperature compound) before cryogenic sectioning improves section quality.

[0058] As mentioned in the background section, existing embedding molds are difficult to meet the needs of embedding tissues of different sizes. To improve this situation, this application has made many improvements to the structure of existing embedding molds, which not only meet the needs of embedding tissues of different sizes, but also solve the problems of tissue embedding angle and section orientation.

[0059] In a first typical embodiment of this application, an embedding mold is provided. The mold is a square funnel shape with a closed bottom, and the inner wall of the mold is marked with graduation lines. By setting the mold to a square funnel shape with a closed bottom and the opening gradually increasing from bottom to top, it is convenient to accommodate tissues of different sizes, thus improving the applicability and flexibility of the mold. The graduation lines 11 marked on the inner wall of the mold facilitate the pre-laying of embedding agent at specific graduation positions according to the size of the tissue to be embedded during the embedding process. This also helps to keep the tissue material in the obtained tissue embedding block in a balanced rather than tilted state, which is beneficial for obtaining complete sections or sections with a specific plane.

[0060] It should be noted that a closed-bottom square funnel shape refers to a shape where the bottom surface at the funnel opening is closed, and the opening gradually increases in size upwards, thus enabling the embedding of tissues of various sizes. Therefore, the specific shape of the closed bottom surface is not particularly limited. For example, it can be any shape such as circular, rectangular, rhomboid, or triangular.

[0061] To further reduce the manufacturing difficulty of the mold, improve the ease of its preparation, and enhance its subsequent use, in a preferred embodiment, the mold includes a square bottom surface and four trapezoidal side surfaces connected to each side of the square bottom surface. The four trapezoidal side surfaces are connected in sequence to form a square funnel shape, and a scale line 11 is marked on the inner wall of any one of the trapezoidal side surfaces.

[0062] It should be noted that the base of this square can be either rectangular or square. Correspondingly, the lengths of the base sides of the trapezoid can be equal or unequal.

[0063] In a preferred embodiment, the area of ​​the square base is (0.5–3) cm × (0.5–3) cm. Specifically, it can be reasonably set according to actual needs, such as 0.5 cm × 0.5 cm, 1.0 cm × 0.5 cm, 0.6 cm × 0.8 cm, 1.0 cm × 1.0 cm, 1.0 cm × 1.5 cm, 2.0 cm × 1.0 cm, 1.0 cm × 3.0 cm, 2.0 cm × 3.0 cm, and 3.0 cm × 3.0 cm. By setting various different specifications, the applicability of this mold is further broadened.

[0064] In the aforementioned mold, the scale lines are used to mark the maximum size of the tissue to be embedded that can be accommodated by different planes parallel to the bottom surface. Therefore, the specific values ​​of the scale lines can be reasonably set according to different specifications. In a preferred embodiment of this application, the scale lines are multiple straight lines set on the inner wall of the trapezoidal side surface and parallel to the connected edges of the trapezoidal side surface (the position of each straight line indicates the height of the plane containing that line from the bottom surface of the embedding mold, and the length of each straight line also indicates, to some extent, the area of ​​the upper surface of the embedded material that can be accommodated). When the area of ​​the square bottom surface is less than 1.0cm × 1.0cm, the lengths of the multiple straight lines are 1.0cm, 1.5cm, 2cm, 2.5cm, and 3cm, respectively.

[0065] Accordingly, in a preferred embodiment, when the square base is a square, the areas of the planes containing each scale line are 1.0cm×1.0cm, 1.5cm×1.5cm, 2cm×2cm, 2.5cm×2.5cm, and 3cm×3cm, respectively.

[0066] In addition, the lengths of multiple straight lines can be refined (for example, the lengths of multiple straight lines can be 1.0cm, 1.1cm, 1.2cm, 1.4cm, 1.5cm, 1.6cm, 1.7cm, 1.8cm, 1.9cm, 2.0cm, 2.1cm, 2.2cm, 2.3cm, 2.4cm, 2.5cm, 2.6cm, 2.7cm, 2.8cm, 2.9cm, and 3.0cm respectively), or made coarser.

[0067] Based on the above-mentioned mold structure improvement, in order to further improve the ability of tissue to maintain a balanced state rather than an arbitrary state (including a tilted state) in the embedding agent, in a preferred embodiment of this application, the mold further includes a level 12, which is disposed on the outer wall of any one or more of the four trapezoidal sides; more preferably, the level is detachably disposed on the outer wall of the trapezoidal side using adhesive tape.

[0068] The specific shape and structure of the level are not limited. Any level that can be installed on the outer wall of the mold to ensure that the mold is placed horizontally, thereby ensuring that the embedding agent is placed horizontally, and further, that the tissue to be embedded on the embedding agent also remains relatively horizontal, is applicable to this application. In a preferred embodiment of this application, the level is a cylinder with a built-in groove filled with water. The levelness of the groove is used to characterize the horizontality of the mold placement.

[0069] To further facilitate the removal of the tissue embedding block from the mold with the aforementioned improved structure, in a preferred embodiment of this application, any one or more of the four trapezoidal sides are detachably connected by snap-fit ​​13. Preferably, the snap-fit ​​13 is located at the top of the side connecting each trapezoidal side to the adjacent trapezoidal side. During embedding, the snap-fit ​​locks in place, forming a square funnel-shaped mold with a closed bottom. After embedding is completed, the snap-fit ​​is opened, revealing the tissue embedding block, making its removal very convenient.

[0070] In order to further improve the orientation of the embedded tissue block itself so that it can be sliced ​​by holding a predetermined angle or plane during subsequent slicing, in a preferred embodiment of this application, the mold also has an embedding direction marking angle 14, which is formed by a plane covering the interior angle formed by any two adjacent trapezoidal sides, and the plane is connected to the square bottom surface.

[0071] The embedding mold of this application can be made of various materials, including but not limited to PVC, PC, PE or PP.

[0072] In a second typical embodiment of this application, a cutting and embedding kit is provided, the kit including an embedding mold, which is any of the embedding molds described above.

[0073] In a third typical embodiment of this application, the application of any of the above-mentioned embedding molds in tissue section embedding is provided, particularly in cryosection embedding. This not only meets the embedding requirements of tissues of different sizes but also maximizes the directional marking of the embedded tissue block and / or keeps the tissue material in the embedded tissue block horizontal, ensuring that the tissue slices obtained during subsequent sectioning are on a single plane, thereby maximizing the accuracy of experimental data.

[0074] In a fourth typical embodiment of this application, a cryo-embedding method is provided, which includes embedding using any of the above-described embedding molds. Using the molds of this application for embedding can meet the embedding requirements of tissues of different sizes, and can also maximize the directional marking of the embedded tissue block, and / or keep the tissue material in the embedded tissue block horizontal, so that the tissue slices obtained during subsequent sectioning are on a single plane, thereby maximizing the accuracy of experimental data.

[0075] In another preferred embodiment, the embedding method includes: laying a bottom layer of OCT at the bottom of a mold up to the graduation line corresponding to the length of the tissue to be embedded; transferring the mold containing the bottom layer of OCT to crushed dry ice; placing the tissue to be embedded in the mold containing the bottom layer of OCT; covering the tissue to be embedded with an upper layer of OCT; and removing the tissue embedding block after the upper layer of OCT has solidified to form a tissue embedding block.

[0076] In the preferred embodiments described above, not only can the appropriate amount of embedding agent be provided to the tissue of a specific size through the scale lines to keep the tissue relatively horizontal, but the mold can also be placed on crushed dry ice to achieve cryo-embedding without the use of toxic organic substances.

[0077] In some preferred embodiments, when transferring the mold containing the underlying OCT onto crushed dry ice, the mold is kept balanced using a level on the mold. Further utilizing the level to maintain the mold's horizontal placement further improves the levelness of the tissue material to be embedded in the final embedded tissue block.

[0078] In some preferred embodiments, the tissue embedding block is removed by opening the clips on the mold, which makes removal easier and prevents damage to the embedded tissue block.

[0079] In some other preferred embodiments, the tissue embedding block has a directional marker 14 to facilitate adjustment of the section angle or direction during subsequent sectioning.

[0080] In a fifth typical embodiment of this application, a method for cryosectioning tissue is provided. This method includes placing cryoembedded tissue on a cryostat for slicing, wherein the cryoembedded tissue is obtained using any of the aforementioned tissue cryoembedded methods. Preferably, the tissue is adjusted to a predetermined direction according to the marked angle on the cryostat and then fixed to the cryostat head for slicing, resulting in directional slices.

[0081] The beneficial effects of this application will be further illustrated below with reference to specific embodiments.

[0082] Using this application Figure 2 The basic steps for embedding and sectioning using the embedding mold shown are as follows:

[0083] 1) Measure the tissue size; 2) Lay a layer of OCT at the bottom of the embedding mold up to the scale line corresponding to the tissue size; 3) Transfer the mold to crushed dry ice, ensuring it is placed in a balanced position; 4) Transfer the tissue into the mold; 5) Cover the tissue with a layer of OCT and wait for it to solidify; 6) Open the clips on both sides of the mold to remove the tissue embedding block; 8) The tissue embedding block has marked corners to identify the tissue embedding direction; 9) Fix the embedded tissue to the frozen head according to the marked corners, ready for sectioning; 10) Set the sectioning temperature; 11) Finally, obtain directional sections.

[0084] Example 1: Tissue embedding and sectioning:

[0085] 1) Crush the dry ice into powder and place it in a container (15cm long, 10cm wide, and 10cm deep);

[0086] 2) Place the OCT bottle upside down on ice (not dry ice), bury the bottle opening in crushed ice, and pre-cool for 10 minutes;

[0087] 3) Place a ruler at the bottom of a petri dish placed on ice, and place the mouse brain from the cryopreservation tube into the petri dish to measure the size of the mouse brain (length approximately 1.3 cm, thickness approximately 0.8 cm);

[0088] 4) Take out the OCT bottle while it is still upside down, unscrew the cap, and lay a layer of OCT on the bottom of the embedding tool up to 1.5cm from the mark;

[0089] 5) If air bubbles are observed in the OCT sample, remove them using a 1000ml pipette;

[0090] 6) After the OCT is laid flat, transfer the embedding tool to the crushed dry ice and place the bottom of the embedding tool in the dry ice powder. At the same time, observe the level and adjust its position to ensure that the tool is placed in a balanced position.

[0091] 7) After the OCT in the embedding tool has solidified, place another 35mm culture dish on ice and pour in about 1ml of OCT. Use tweezers to hold the mouse brain and gently roll it around once. Then place it in the embedding tool, adjust the orientation, and cover the mouse brain with a layer of OCT. In this step, it is important to ensure that the OCT completely covers the mouse brain. There should be no air bubbles around the tissue to be cut. At the same time, add a lid on top and place dry ice on the lid to accelerate the freezing speed.

[0092] 8) After the OCT covering solidifies, remove the embedding tool from the dry ice and quickly open the clips on both sides of the mold. Use tweezers to remove the embedding block and put it into a bag for storage at -80 degrees Celsius or proceed to the next step of sectioning (at this time, the embedding block has a marked corner to identify the direction).

[0093] 9) Place the OCT block embedded with mouse brain tissue on a frozen sample holder covered with a layer of liquid OCT, adjust it to the desired orientation according to the corner markings on the embedded block, and then perform rapid freezing fixation;

[0094] 10) After the OCT of the tissue sample is completely fixed, fix the tissue block onto the frozen head and test the section temperature conditions (as shown in the table below). According to the flatness of the section, the chamber temperature of -20°C and the frozen head temperature of -10°C and the chamber temperature of -26°C and the frozen head temperature of -16°C are more suitable for mouse brain tissue sections.

[0095] -25 -25 same Wrinkled + Fragile -20 -20 same Flattening + Rolling -20 -15 5 Flat + Severely Curled -20 -10 10 Flat and non-curling -26 -16 10 Flat and non-curling -25 -10 15 Flattening + Rolling -16 -26 10 Wrinkled + Curled + Fragile -10 -20 10 Severe wrinkles

[0096] 11) Mount the complete tissue sections onto a glass slide and perform HE staining (operate in a fume hood);

[0097] a) Experimental preparation: First, dispense the reagents required for the experiment into 50ml centrifuge tubes, each tube containing no less than 30ml, and arrange them in the order of the operation steps for subsequent operations (note that the ultrapure water used in the water washing step is disposable).

[0098] b) Fixation and washing: Acetone was pre-cooled to 4°C, and then the slide with the slice attached (hereinafter referred to as slide) was placed in a centrifuge tube containing acetone for 10 min to fix it. Then it was transferred to a centrifuge tube containing ultrapure water and washed for 2 s.

[0099] c) Hematoxylin staining: Transfer the slide to a centrifuge tube containing hematoxylin and stain for 3-5 minutes; after the staining time is over, transfer the slide to a centrifuge tube containing ultrapure water and wash for 2 seconds.

[0100] d) Differentiation: Transfer the slide to the differentiation solution and differentiate for 2 seconds (differentiation solution: take 33 ml of anhydrous ethanol into the BD tube, add 1.73 ml of H2O and 0.35 ml of HCl to prepare 35 ml of differentiation solution in one tube), place the slide in a vertical slide container, and run tap water to blue for 8 minutes.

[0101] e) Eosin staining: Transfer the slide to a centrifuge tube containing eosin and stain for 2 seconds (this step needs to be done quickly, as too long a time will result in the tissue section being too dark).

[0102] f) Washing and dehydration: Transfer the glass slide to a centrifuge tube containing ultrapure water and wash for 2 seconds, then continuously transfer it to centrifuge tubes containing 80%, 95% ethanol and anhydrous ethanol respectively for 2 seconds each.

[0103] g) Tissue transparency: The slide was continuously transferred in three tubes (I, II, III) of xylene for 2 seconds per tube;

[0104] h) Fixation: Transfer the slide to lint-free paper (with the slide side up) and let it dry; apply a drop of neutral resin to the slide and cover it with a coverslip to seal it;

[0105] 12) Observe the slide under a microscope and take pictures.

[0106] As can be seen from the above embodiments, commercially available embedding molds are generally of fixed size and cannot be adjusted according to tissue size. Furthermore, their small size makes them suitable only for embedding small tissues, failing to completely cover larger tissues. While embedding in petri dishes offers the advantage of complete coverage for larger tissues, it requires extensive modification during sectioning, making it relatively cumbersome. Both methods can lead to sample tilting due to unbalanced mold placement during embedding, resulting in tissue slices not being on the same plane and distorting experimental data. Additionally, both methods are inconvenient to disassemble. This application combines the advantages of both methods while overcoming their disadvantages. Simultaneously, embedding conditions were tested and optimal embedding conditions were determined; the toxicity to operators caused by isopentane in traditional cryo-embedding methods is reduced.

[0107] 1) Strong compatibility with sample size: Traditional embedding tools are only suitable for embedding samples of one size. However, the mold invented in this method adopts a three-dimensional trapezoidal shape, with a base area of ​​0.5cm × 0.5cm, gradually expanding from the bottom to the top. Scale lines are marked on one side of the mold, indicating sizes of 1.5cm × 1.5cm, 2cm × 2cm, 2.5cm × 2.5cm, and 3cm × 3cm. The same embedding tool can accommodate embedding tissues of various diameters from 0.5cm to 3cm. Figure 2 ).

[0108] 2) Reduced operation time: When embedding in cell culture dishes, a large area of ​​OCT around the sample needs to be modified. However, with this method, there is no need to modify the OCT around the embedded sample, reducing operation time and the possibility of damage to the tissue sample during modification.

[0109] 3) Increased sectioning success rate: Traditional embedding methods cannot ensure uniform thickness of the obtained tissue OCT blocks. This method, however, uses a level on the embedding tool to determine if the mold is balanced, ensuring the sample is on a uniformly thick OCT plane during embedding. This reduces the probability that uneven embedding reagent thickness will result in tissue sections that are not on the cross-sections required by the experimental design. Figure 1 right).

[0110] 4) Easy embedding and removal: Traditional methods using commercially available embedding cassettes or cell culture dishes suitable for large tissue samples require waiting for the outer OCT layer of the embedding block to slightly melt before using tweezers to pry it out. The embedding mold of this invention can be opened directly from both sides to remove the embedding block, saving time and effort and making the operation more convenient.

[0111] 5) Facilitates identification of sample embedding orientation: In traditional methods, the tissue embedding orientation can only be marked on the sample embedding tool. This makes it very difficult to determine the orientation when the sample is removed for subsequent operations, increasing the difficulty of the experiment. However, in the method of this invention, the tissue embedding orientation can be identified at any stage of the operation by marking the corner of the OCT block embedded in the tissue sample, which helps to increase the success rate of the experiment.

[0112] 6) Avoid reagent toxicity: Cryo-embedding using crushed ice combined with dry ice powder avoids the toxicity associated with isopentane embedding. In the early stages of this invention, reagent and consumable combinations for preservation, transportation, and embedding of fresh samples within 30 minutes of in vitro exposure were tested. Through several rounds of testing, it was determined that tissues transported on crushed ice within 30 minutes of in vitro exposure, and embedding on dry ice powder to increase the contact area between the tissue and dry ice for faster cooling, were most effective. Embedding within 30 minutes of in vitro exposure is also crucial for optimal results, avoiding the toxicity associated with isopentane cryo-embedding using liquid nitrogen combined with isopentane, and eliminating the need for a fume hood, thus reducing operational complexity. Figure 3 (Explanation of HE staining effect on sections).

[0113] It should be noted that the embedding mold of this application is not only applicable to tissue embedding sections, but also applicable to cell embedding sections according to actual needs.

[0114] The above are merely preferred embodiments of the present invention and are not intended to limit 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 scope of protection of the present invention.

Claims

1. An embedding mold, characterized in that, The embedding mold is a square funnel shape with a closed bottom, and the inner wall of the embedding mold is marked with scale lines. The embedding mold includes a square bottom surface and four trapezoidal side surfaces connected to each side of the square bottom surface. The four trapezoidal side surfaces are connected in sequence to form the square funnel shape. The scale line is marked on the inner wall of any one of the trapezoidal side surfaces. The material of the mold is selected from PVC, PC, PE or PP.

2. The embedding mold according to claim 1, characterized in that, The area of ​​the square base is (0.5~3)cm × (0.5~3)cm.

3. The embedding mold according to claim 1, characterized in that, The scale lines are multiple straight lines set on the inner wall of the trapezoidal side and parallel to the connected sides of the trapezoidal side, with the lengths of the multiple straight lines being 1.0cm, 1.5cm, 2cm, 2.5cm and 3cm respectively.

4. The embedding mold according to claim 3, characterized in that, The square base is square, and the areas of the planes containing the scale lines are 1.0cm×1.0cm, 1.5cm×1.5cm, 2cm×2cm, 2.5cm×2.5cm and 3cm×3cm, respectively.

5. The embedding mold according to any one of claims 1 to 4, characterized in that, The embedding mold also includes a level, which is set on the outer wall of any one or more of the four trapezoidal sides.

6. The embedding mold according to claim 5, characterized in that, The level is detachably mounted on the outer wall of the trapezoidal side using adhesive tape.

7. The embedding mold according to claim 5, characterized in that, Any one or more of the four trapezoidal sides are detachably connected by snap-fit.

8. The embedding mold according to claim 7, characterized in that, The buckle is located at the top of the side of each trapezoidal side that connects to the side of the adjacent trapezoidal side.

9. The embedding mold according to claim 5, characterized in that, The embedding mold also has an embedding direction marking angle, which is formed by a plane that covers the interior angle formed by any two adjacent trapezoidal sides, and the plane is connected to the square bottom surface.

10. An embedding kit, characterized in that, The kit includes an embedding mold, which is the embedding mold according to any one of claims 1 to 9.

11. The application of the embedding mold according to any one of claims 1 to 9 in embedding sections.

12. The application according to claim 11, characterized in that, The application is cryoembedded sectioning.

13. A cryo-embedding method, characterized in that, The embedding method includes embedding using the embedding mold described in any one of claims 1 to 9.

14. The embedding method according to claim 13, characterized in that, The embedding method includes: A bottom layer of OCT is laid at the bottom of the mold up to the graduation line corresponding to the length of the tissue to be embedded; Transfer the mold containing the bottom layer OCT onto crushed dry ice; The tissue to be embedded is placed in the mold containing the underlying OCT; Cover the tissue to be embedded with an upper layer of OCT; After the upper OCT layer solidifies, a tissue embedding block is formed and the tissue embedding block is removed.

15. The embedding method according to claim 14, characterized in that, When transferring the mold containing the bottom layer of OCT onto crushed dry ice, the mold is kept balanced by using a level on the mold.

16. The embedding method according to claim 15, characterized in that, Open the clips on the mold to remove the tissue embedding block.

17. The embedding method according to claim 15, characterized in that, The tissue embedding block has directional markers.

18. A method for cryosectioning, characterized in that, The slicing method includes placing the cryoembedded tissue on a cryostat for slicing, wherein the cryoembedded tissue is obtained by embedding using the embedding method described in any one of claims 13 to 17.

19. The slicing method according to claim 18, characterized in that, After adjusting the frozen tissue embedding block to the predetermined direction according to the marked angle, it is fixed to the freezing head of the cryostat for sectioning, resulting in directional sections.

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