A plastic embedding agent and embedding method suitable for bulk sample thick sectioning

Abstract

The application discloses a plastic embedding agent Big Cut resin, which is composed of a crosslinking agent and a non-reactive liquid auxiliary agent in a mass ratio of 3:7 to 7:3. The application further discloses a preparation method of the plastic embedding agent, which comprises the steps of mixing the crosslinking agent and the non-reactive liquid auxiliary agent in the mass percentage to obtain a mixture, and adding an initiator into the mixture to obtain the Big Cut resin. The application further discloses a plastic embedding method suitable for thick cutting of a large-volume sample, which embeds a biological tissue sample by using the Big Cut resin. The embedding method can realize uniform and stable polymerization of the large-volume resin, improve the cutting performance of the sample after embedding, meet the thick cutting requirement of an fMOST system, has the advantages of transparent depth imaging and adjustable refractive index, and realizes high-throughput imaging.

Description

Technical Field

[0001] This invention belongs to the field of plastic embedding technology, specifically, it relates to a plastic embedding agent and embedding method suitable for thick cutting of large volume samples. Background Technology

[0002] In the biomedical field, the three-dimensional reconstruction of large-volume intact tissue structures is of vital scientific and clinical significance for a deeper understanding of the physiological and pathological functions of tissues and organs, precise analysis of the mechanisms of disease development, and the implementation of new drug research and precision medicine technologies. It is also a core technological support for achieving cross-scale structural analysis from microscopic cells to macroscopic organs. The combination of optical microscopy and tissue transparency techniques can overcome the depth limitations of traditional imaging, enabling the acquisition of high-resolution, high-signal-to-noise-ratio structural information from large-volume intact samples, becoming the mainstream direction of current biomedical three-dimensional imaging research.

[0003] Resin embedding is a common sample preparation method in optical microscopy to ensure sample morphology integrity and improve imaging stability. However, existing large-volume resin transparent embedding techniques still face many insurmountable bottlenecks, failing to meet the practical needs of scientific research and clinical applications. On the one hand, large-volume resin systems are difficult to achieve stable and uniform polymerization during curing, leading to problems such as uneven hardness, excessive internal stress, and cracking deformation within the embedded block. This cannot fully meet the continuous cutting requirements of high-precision imaging systems, easily causing sample damage and imaging tomography. On the other hand, the layer-by-layer cutting and imaging process for large-volume samples is time-consuming, and the resulting massive amounts of three-dimensional data exacerbate the pressure on storage, processing, and analysis, significantly reducing research efficiency.

[0004] Due to common technical challenges such as resin penetration difficulties and uneven polymerization in large-volume samples, current research rarely applies resin embedding to the preparation of samples with dimensions of 100 cubic centimeters, which greatly limits the advancement of cross-scale tissue structure research. For ultra-large-volume samples, the prominent problem with ultra-thin cutting in traditional resin embedding methods is the long imaging time and huge data volume, making it difficult to achieve rapid and efficient imaging. For three-dimensional microscopy, the XY direction can expand the field of view and improve the throughput of planar imaging by using low-magnification high numerical aperture objectives (such as Olympus PLN 10X, NA=0.6); however, the Z direction is limited by traditional acrylic resins (such as GMA, Lowicyl...). ® Due to the mechanical properties of HM20, it can only achieve ultra-thin continuous slices with a thickness of micrometer or submicrometer. It cannot reasonably reduce the sampling interval or optimize the cutting thickness, resulting in a significant increase in the cutting-imaging cycle and an exponential increase in the overall imaging time, which seriously restricts the large-scale application of three-dimensional reconstruction of large-volume samples.

[0005] Therefore, a new plastic embedding agent and embedding method are needed to overcome the shortcomings of the existing technology and break through the technical barriers of resin embedding and high-efficiency imaging of large-volume tissue samples. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a plastic embedding agent and embedding method suitable for thick cutting of large-volume samples.

[0007] Specifically, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a plastic embedding agent, wherein the embedding agent is Big Cut resin, and the resin is composed of a crosslinking agent and a non-reactive liquid additive in a mass ratio of 3:7 to 7:3.

[0009] In one or more embodiments, the crosslinking agent is triethylene glycol dimethacrylate (TEGDMA) or 1,4-butanediol dimethacrylate (BDDMA); the non-reactive liquid additive is benzyl benzoate (BB) or methyl salicylate (MeS).

[0010] In one or more embodiments, the embedding agent further comprises a resin formed from a low-refractive-index resin monomer, said low-refractive-index resin monomer being one or more of 2,2,3,3-tetrafluoropropyl methacrylate, benzyl formate, benzyl alcohol, and methyl anisinate.

[0011] In a second aspect, the present invention provides a method for preparing the plastic embedding agent according to any one of the above claims, the method comprising the steps of mixing a crosslinking agent and a non-reactive liquid additive in the mass ratio to obtain a mixture, and adding an initiator to the mixture to obtain a Big Cut resin.

[0012] In one or more embodiments, optionally, a low-refractive-index resin monomer is added to the mixture along with the initiator.

[0013] In one or more embodiments, the initiator is azobisisobutyronitrile, which accounts for 0.03%-0.1% of the total mass of Big Cut resin.

[0014] In one or more embodiments, if a low-refractive-index resin monomer is not added to the mixture, the crosslinking agent accounts for 30%-70% of the total mass of the Big Cut resin, and the non-reactive liquid additive accounts for 30%-70% of the total mass of the Big Cut resin.

[0015] In one or more embodiments, if a low-refractive-index resin monomer is added to the mixture, the low-refractive-index resin monomer accounts for 0%-30% of the total mass of the Big Cut resin.

[0016] In one or more embodiments, the preparation method further includes a pretreatment step of the crosslinking agent, wherein the pretreatment step is: filtering the crosslinking agent using a glass chromatography column, inserting a small amount of degreased cotton at the outlet of the chromatography column, adding alkaline alumina powder, and filtering out the polymerization inhibitor in the crosslinking agent.

[0017] Thirdly, the present invention provides a plastic embedding method suitable for thick cutting of large-volume samples, the plastic embedding method comprising the step of embedding large-volume biological tissue samples with the plastic embedding agent described in any of the above claims or the plastic embedding agent prepared by any of the above claims.

[0018] In one or more embodiments, the plastic embedding method includes the following steps:

[0019] (1) Pretreatment: Decalcification and deesterification reagents were used to decalcify and deesterify the biological samples respectively. Then, the fixed biological tissue samples were rinsed multiple times with buffer solution, with the buffer solution being changed every 3-4 hours.

[0020] (2) Gradient dehydration: The pretreated biological tissue samples were placed in 50% ethanol pre-cooled at 4°C overnight, and then placed in 75% ethanol, 95% ethanol and anhydrous ethanol pre-cooled at 4°C for gradient dehydration for 6-8 hours at each gradient. After that, they were placed in anhydrous ethanol overnight to completely dehydrate the biological tissue samples.

[0021] (3) Gradient permeation: The plastic embedding agent Big Cut resin was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% Big Cut resin pre-cooled at 4°C overnight, and then placed in 75% and 100% Big Cut resin for gradient permeation, each gradient lasting 6-12 hours; finally, the biological tissue samples were placed in 100% Big Cut resin overnight, then placed in 100% Big Cut resin for 24 hours, and then placed in 100% Big Cut resin for 48 hours to allow the Big Cut resin to fully permeate the biological tissue samples;

[0022] (4) Polymerization and embedding: The infiltrated biological tissue sample is placed in a mold, and then 100% Big Cut resin is slowly added to the mold to fill it. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

[0023] In one or more embodiments, the decalcification reagent and the deesterification reagent in step (1) are both aqueous solutions; wherein the decalcification reagent is a 10% (w / w) disodium ethylenediaminetetraacetate (EDTA-2Na) solution; wherein the deesterification reagent is prepared from 0.05% (w / w) PI, 10% (w / w) CHAPS, 2% (w / w) Triton X-100, 10% (w / w) DMSO, 1% (w / w) glycine, and 1% (w / w) cyclodextrin; the buffer solution is a 0.01M phosphate buffer; and the biological tissue sample is rinsed three times with the buffer solution.

[0024] In one or more embodiments, the thermal polymerization in step (4) is carried out in an oven at a temperature of 36-45°C for a time of 36-72 hours.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) To address the problem of uneven resin polymerization in large-volume samples, the present invention uses Big Cut resin to achieve uniform and stable polymerization;

[0027] (2) By utilizing the high refractive index and non-reactive properties of non-reactive liquid additives, the thick cutting requirements of the fMOST system can be met. At the same time, it has the advantages of transparent depth imaging and adjustable refractive index, thus realizing high-throughput imaging. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the polymerization principle of Big Cut, an encapsulating agent without the addition of low-refractive-index resin monomers.

[0029] Figure 2 This is a schematic diagram of the polymerization principle of Big Cut, an encapsulating agent containing low-refractive-index resin monomers.

[0030] Figure 3 This is a picture of the Big Cut embedding agent polymer.

[0031] Figure 4 This is a Big Cut thickness test chart for embedding agents.

[0032] Figure 5 This is a detailed image of mouse brain LBA1 embedded in Big Cut resin and stained with immunofluorescence on fMOST; where A is a schematic diagram of fMOST imaging, B is the three-dimensional imaging result of mouse brain embedded in Big Cut resin, and CH is the magnified image marked in B. Detailed Implementation

[0033] In this field, to improve the imaging efficiency of large-volume samples, transparent embedding is performed on the samples during the sample preparation stage, thereby increasing the depth of a single imaging session and shortening the imaging time. This invention uses a crosslinking agent as the main component of the resin system and selects a non-reactive liquid additive that matches the tissue's refractive index to achieve transparent embedding.

[0034] In this invention, Big Cut resin constructs a three-dimensional cross-linked network during polymerization, thereby enhancing the mechanical strength and stability of the resin; the non-reactive liquid additive, although not participating in the chemical reaction, has a refractive index that matches the tissue, which can increase the transparency of the sample, reduce light scattering, and improve imaging quality.

[0035] In this invention, the embedding agent is Big Cut resin, whose main components are crosslinking agents and non-reactive liquid additives in a mass ratio of 3:7 to 7:3, such as 3:7, 1:1, 3:2, 3:4, 7:3, etc.

[0036] In this invention, the crosslinking agent is a type of chemical that participates in the resin crosslinking reaction, causing the active functional groups in the resin to connect with each other to form a network structure. The crosslinking agent primarily improves the viscosity and elasticity of the working fluid, and should also possess certain temperature and salt resistance properties depending on the reservoir geological characteristics. In addition, some additives are added to improve the overall performance of the working fluid. To preserve the crosslinking agent for a long time, a small amount of polymerization inhibitor is added; therefore, in the preparation of the encapsulating agent of this invention, the polymerization inhibitor in the crosslinking agent needs to be filtered out first.

[0037] It should be understood that the crosslinking agent described in this invention is common knowledge in the art, also known as a curing agent, hardening agent, ripening agent, etc. It can transform linear or slightly branched macromolecules into a three-dimensional network structure, thereby improving properties such as strength, heat resistance, abrasion resistance, and solvent resistance. In the art, any chemical substance with the same function can be tried in the preparation of the Big Cut resin described in this invention, such as triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, etc.

[0038] In one or more embodiments, the crosslinking agent is triethylene glycol dimethacrylate, which accounts for 30-70% of the total mass of Big Cut resin, such as 30%, 50%, 65%, 70%, etc.

[0039] In this invention, the non-reactive liquid additive is a small molecule or oligomer functional auxiliary component used in polymer materials, adhesives, coatings, composite materials, and optical resin systems. It is liquid at room temperature and pressure and does not participate in the main polymerization, grafting, or crosslinking chemical bonding reactions of the matrix resin and crosslinking system within the specified processing, curing, molding, and long-term use conditions of the material. It is blended with the system only through physical dissolution, molecular dispersion, and interfacial wetting. It is used to improve processing rheological properties, adjust the physical and mechanical properties of the material, optimize interfacial compatibility, and assist in functional regulation. It is an external functional auxiliary material added to the formulation and does not constitute a covalently bonded structural unit of the three-dimensional network or continuous phase skeleton of the cured polymer.

[0040] It should be understood that the non-reactive liquid additives described in this invention are common knowledge in the art. They are low-volatility, resin-compatible liquid substances that exist in small molecule liquid form and play an auxiliary role. In the art, any chemical substance with the same function can be tried in the preparation of the Big Cut resin described in this invention, such as benzyl benzoate (BB) or methyl salicylate (MeS).

[0041] In one or more embodiments, the non-reactive liquid additive is methyl salicylate, which accounts for 30%-70% of the total mass of Big Cut resin, such as 30%, 40%, 50%, 70%, etc.

[0042] In this invention, the introduction of the low-refractive-index resin monomer is to reduce the refractive index of the embedding agent, thereby achieving high-throughput imaging when combined with the imaging system. In the art, any chemical substance with the same function can be used in the preparation of the Big Cut resin described in this invention, such as 2,2,3,3-tetrafluoropropyl methacrylate, benzyl formate, benzyl alcohol, and methyl anisinate. Those skilled in the art can add one or more low-refractive-index resin monomers according to actual needs to adjust the refractive index of the embedding agent.

[0043] In one or more embodiments, the low refractive index resin monomer is 2,2,3,3-tetrafluoropropyl methacrylate, which accounts for 0%-30% of the total mass of Big Cut resin, such as 10%, 20%, 30%, etc.

[0044] It should be understood that the initiators described in this invention are common knowledge in the art. An initiator is a substance capable of initiating a polymerization reaction of monomers. Unsaturated monomer polymerization active centers include free radicals, anionic compounds, cationic compounds, and coordination compounds. In the adhesive industry, the free radical type is the most widely used, exhibiting unique chemical activity. Under the influence of heat or light, it undergoes homolytic cleavage of covalent bonds to generate two free radicals, which can initiate a polymerization reaction. In the art, any chemical substance with similar activity can be attempted to be used in the preparation of the Big Cut resin described in this invention, such as azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (AIHHNnitrile).

[0045] In one or more embodiments, the initiator is azobisisobutyronitrile, which accounts for 0.03%-0.2% of the total mass of Big Cut resin, such as 0.03%, 0.05%, 0.06%, 0.1%, 0.2%, etc.

[0046] The Big Cut resin, a plastic embedding agent described in this invention, can be used for plastic embedding of large-volume biological tissue samples.

[0047] It should be understood that, in this field, three-dimensional reconstruction of large-volume intact tissue structures is of great significance for in-depth understanding of organ function and research on disease mechanisms. By fully fusing pretreated large-volume biological tissue with resin polymers and polymerizing under certain conditions, the polymerized sample exhibits high hardness, enabling the slicing of biological tissues and better application in optical imaging. Through continuous research and exploration, the inventors of this invention have discovered that Big Cut resin, after plastic embedding of large-volume biological tissue samples, can achieve high-throughput imaging. The polymerization principle of the Big Cut resin is as follows... Figure 1 and Figure 2 As shown, where Figure 1 This is a schematic diagram illustrating the polymerization process without the addition of low-refractive-index resin monomers. Figure 2 This diagram illustrates the polymerization principle of adding low-refractive-index resin monomers. Therefore, Big Cut resin has wide applications in this field and is of great significance for studying biological tissue structures and exploring disease mechanisms.

[0048] The present invention also includes a plastic embedding method suitable for thick cutting of large volume samples, the plastic embedding method comprising the step of embedding large volume biological tissue samples with the plastic embedding agent described above or the plastic embedding agent prepared by any of the above methods.

[0049] In one or more embodiments, the plastic embedding method includes the following steps:

[0050] (1) Pretreatment: Decalcification and deesterification reagents (both aqueous solutions) were used to decalcify and deesterify the biological samples respectively. Then, the fixed biological tissue samples were rinsed multiple times with buffer solution, with the buffer solution being changed every 3-4 hours.

[0051] (2) Gradient dehydration: The pretreated biological tissue samples were placed in 50% ethanol pre-cooled at 4°C overnight, and then placed in 75% ethanol, 95% ethanol and anhydrous ethanol pre-cooled at 4°C for gradient dehydration for 6-8 hours at each gradient. After that, they were placed in anhydrous ethanol overnight to completely dehydrate the biological tissue samples.

[0052] (3) Gradient permeation: The plastic embedding agent Big Cut resin was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% Big Cut resin pre-cooled at 4°C overnight, and then placed in 75% and 100% Big Cut resin for gradient permeation, each gradient lasting 6-12 hours; finally, the biological tissue samples were placed in 100% Big Cut resin overnight, then placed in 100% Big Cut resin for 24 hours, and then placed in 100% Big Cut resin for 48 hours to allow the Big Cut resin to fully permeate the biological tissue samples;

[0053] (4) Polymerization and embedding: The infiltrated biological tissue sample is placed in a mold, and then 100% Big Cut resin is slowly added to the mold to fill it. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

[0054] In one or more embodiments, the decalcification reagent in step (1) is a 10% (w / w) disodium ethylenediaminetetraacetate (EDTA-2Na) solution; the ester removal reagent is prepared from 0.05% (w / w) PI, 10% (w / w) CHAPS, 2% (w / w) Triton X-100, 10% (w / w) DMSO, 1% (w / w) glycine, and 1% (w / w) cyclodextrin; the buffer solution is a 0.01M phosphate buffer; the biological tissue sample is rinsed 2-4 times with the buffer solution, such as 3 times.

[0055] In one or more embodiments, the thermal polymerization in step (4) is carried out in an oven at a temperature of 36-45°C for a time of 36-72 hours.

[0056] It should be understood that the biological tissue samples described in this invention can be human or animal tissues, and the specific tissue is not limited. For example, they can be tissues from different parts of the body, such as all or part of the brain, heart, trunk, spinal cord, etc. These biological tissue samples, when embedded with the embedding agent described in this invention, can achieve uniform aggregation of the samples.

[0057] It should be understood that, in order to verify the embedding effect of Big Cut resin, animal tissues can be used for experiments, such as using fluorescently labeled mouse tissue to verify the effect of transparent embedding.

[0058] As an example, such as Figure 3 The image shown is a physical representation of a mouse whole-body embedded using Big Cut resin. Of course, for different biological tissue samples, minor steps or reagent adjustments may be involved in the embedding process; these adjustments are routine procedures for those skilled in the art.

[0059] It should be understood that the plastic embedding method described in this invention is not fixed. Those skilled in the art can make appropriate adjustments to the steps or specific parameters according to different animal tissues, etc., to adapt to different studies. For example, after step (3), the animal tissue can be acidified. For example, the animal tissue can be placed in 100% Big Cut resin containing 25 μl / 10 ml acetic acid for 12-14 hours to acidify the animal tissue; and then subsequent steps can be carried out.

[0060] Example

[0061] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0065] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0066] In the following examples, the crosslinking agent triethylene glycol dimethacrylate was purchased from Macklin; the non-reactive liquid additive methyl salicylate was purchased from Macklin; the low refractive index resin monomer 2,2,3,3-tetrafluoropropyl methacrylate was purchased from Macklin; and the initiator azobisisoheptanenitrile was purchased from Adamas.

[0067] Example 1: A method for preparing a plastic embedding agent, Big Cut resin

[0068] A plastic embedding agent, Big Cut resin, wherein the monomers of the resin are composed of a crosslinking agent and a non-reactive liquid additive in a 3:2 mass ratio.

[0069] The preparation steps of the above-mentioned Big Cut resin are as follows: the crosslinking agent is filtered using a glass chromatography column, a small amount of degreased cotton is inserted into the outlet of the chromatography column, alkaline alumina powder is added, the polymerization inhibitor in the resin is filtered out, and then the crosslinking agent and non-reactive liquid additives are mixed in the specified proportion to obtain a mixture. Then, an initiator is added to react and obtain the Big Cut resin. The crosslinking agent is triethylene glycol dimethacrylate, which accounts for 60% of the total mass of the Big Cut resin; the non-reactive liquid additive is methyl salicylate, which accounts for 40% of the total mass of the Big Cut resin; and the initiator is azobisisoheptanenitrile, which accounts for 0.06% of the total mass of the Big Cut resin.

[0070] Example 2: A method for preparing a plastic embedding agent, Big Cut resin

[0071] A plastic embedding agent, Big Cut resin, wherein the monomers of the resin are composed of a crosslinking agent and a non-reactive liquid additive in a 1:1 mass ratio.

[0072] The preparation steps of the above-mentioned Big Cut resin are as follows: the crosslinking agent is filtered using a glass chromatography column, a small amount of degreased cotton is inserted into the outlet of the chromatography column, alkaline alumina powder is added, the polymerization inhibitor in the resin is filtered out, and then the crosslinking agent and non-reactive liquid additives are mixed in the above proportion to obtain a mixture. Then, an initiator is added to react and obtain the Big Cut resin. The crosslinking agent is 1,4-butanediol dimethacrylate, which accounts for 50% of the total mass of the Big Cut resin; the non-reactive liquid additive is benzyl benzoate, which accounts for 50% of the total mass of the Big Cut resin; and the initiator is azobisisobutyronitrile, which accounts for 0.1% of the total mass of the Big Cut resin.

[0073] Example 3: A method for preparing a plastic embedding agent, Big Cut resin

[0074] A plastic embedding agent, Big Cut resin, wherein the monomers of the resin are composed of a crosslinking agent and a non-reactive liquid additive in a mass ratio of 7:3.

[0075] The preparation steps of the above-mentioned Big Cut resin are as follows: the crosslinking agent is filtered using a glass chromatography column, a small amount of degreased cotton is inserted into the outlet of the chromatography column, alkaline alumina powder is added, the polymerization inhibitor in the resin is filtered out, and then the crosslinking agent and non-reactive liquid additives are mixed in the above proportion to obtain a mixture. Then, an initiator is added to react and obtain the Big Cut resin. The crosslinking agent is triethylene glycol dimethacrylate, which accounts for 70% of the total mass of the Big Cut resin; the non-reactive liquid additive is benzyl benzoate, which accounts for 30% of the total mass of the Big Cut resin; and the initiator is azobisisoheptanenitrile, which accounts for 0.03% of the total mass of the Big Cut resin.

[0076] Example 4: A method for preparing a plastic embedding agent, Big Cut resin

[0077] A plastic embedding agent, Big Cut resin, wherein the monomers of the resin are composed of a crosslinking agent, a non-reactive liquid additive, and a low refractive index resin monomer in a mass ratio of 3:4:3.

[0078] The preparation steps of the above-mentioned Big Cut resin are as follows: the crosslinking agent is filtered using a glass chromatography column, a small amount of degreased cotton is inserted into the outlet of the chromatography column, alkaline alumina powder is added, the polymerization inhibitor in the resin is filtered out, and then the crosslinking agent and non-reactive liquid additives are mixed in the specified proportion to obtain a mixture. Then, a low refractive index resin monomer and an initiator are added to react and obtain the Big Cut resin. The crosslinking agent is triethylene glycol dimethacrylate, which accounts for 30% of the total mass of the Big Cut resin; the non-reactive liquid additive is methyl salicylate, which accounts for 40% of the total mass of the Big Cut resin; the low refractive index resin monomer is 2,2,3,3-tetrafluoropropyl methacrylate, which accounts for 30% of the total mass of the Big Cut resin; and the initiator is azobisisoheptanenitrile, which accounts for 0.06% of the total mass of the Big Cut resin.

[0079] Test Example 1: Cutting Thickness Test of Plastic Embedding Agent Big Cut Resin

[0080] The Big Cut plastic embedding agent prepared in Example 4 was cut to different thicknesses on an imaging system.

[0081] The results are as follows Figure 4 As shown, complete sections can be achieved in the 6-25μm range, indicating that the embedding agent of the present invention can achieve the purpose of thick cutting.

[0082] Application Example 1: Fine imaging of mouse brain LBA1 immunofluorescence staining on fMOST using Big Cut embedded material.

[0083] The biological tissue sample used in this embodiment is a mouse brain, which has been treated with Iba1 immunofluorescence staining.

[0084] (1) Pretreatment: The biological samples were decalcified and deesterified using decalcification reagent and deesterification reagent (both aqueous solutions), respectively. Then, the fixed biological tissue samples were rinsed multiple times with buffer solution, with the buffer solution being changed every 3-4 hours.

[0085] The decalcification reagent was a 10% (w / w) aqueous solution of disodium ethylenediaminetetraacetate (EDTA-2Na); the ester removal reagent was prepared from 0.05% (w / w) PI, 10% (w / w) CHAPS, 2% (w / w) Triton X-100, 10% (w / w) DMSO, 1% (w / w) glycine, and 1% (w / w) cyclodextrin (the remainder being water); the buffer solution was a 0.01M phosphate buffer; the biological tissue sample was rinsed three times with the buffer solution.

[0086] (2) Gradient dehydration: The pretreated biological tissue samples were placed in 50% ethanol pre-cooled at 4°C overnight, and then placed in 75% ethanol, 95% ethanol and anhydrous ethanol pre-cooled at 4°C for gradient dehydration, each gradient lasting 7 hours. After that, the samples were placed in anhydrous ethanol overnight to completely dehydrate the biological tissue samples.

[0087] (3) Gradient permeation: The plastic embedding agent Big Cut resin prepared in Example 1 was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% Big Cut resin pre-cooled at 4°C overnight, and then placed in 75% and 100% Big Cut resin for gradient permeation, each gradient lasting 8 hours; finally, the biological tissue samples were placed in 100% Big Cut resin overnight, then placed in 100% Big Cut resin for 24 hours, and then placed in 100% Big Cut resin for 48 hours to allow the Big Cut resin to fully permeate the biological tissue samples;

[0088] (4) Polymerization and embedding: The permeated biological tissue sample is placed in a 000 gelatin capsule, and then 100% Big Cut resin is slowly added to the 000 gelatin capsule to fill it completely. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample. The thermal polymerization is carried out in an oven at a temperature of 40°C for 50 hours.

[0089] The embedded mouse brain was imaged using the fMOST system in pure water with a precision cut to 1 μm. The resulting immunofluorescence images of mouse brain vessels are shown below. Figure 5 As shown, the Big Cut resin described in this invention can effectively preserve the fluorescence signal of biological tissue samples after embedding them.

[0090] As is known to those skilled in the art, imaging larger samples requires increasing imaging throughput, i.e., increasing sample transparency and reducing resin refractive index, to meet the purpose of thickness-cutting depth imaging in the imaging system. Therefore, in this invention (Application Example 1), adding low-refractive-index resin monomers to reduce the resin refractive index can achieve more efficient depth imaging results.

[0091] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A plastic embedding agent, characterized in that, The embedding agent is Big Cut resin, which is composed of crosslinking agent and non-reactive liquid additive in a mass ratio of 3:7 to 7:

3.

2. The plastic embedding agent as described in claim 1, characterized in that, The crosslinking agent is triethylene glycol dimethacrylate or 1,4-butanediol dimethacrylate; the non-reactive liquid additive is benzyl benzoate or methyl salicylate.

3. The plastic embedding agent as described in claim 1, characterized in that, The embedding agent also includes a resin formed from a low-refractive-index resin monomer, wherein the low-refractive-index resin monomer is one or more of 2,2,3,3-tetrafluoropropyl methacrylate, benzyl formate, benzyl alcohol, and methyl anisinate.

4. A method for preparing the plastic embedding agent according to any one of claims 1-3, characterized in that, The preparation method includes the steps of mixing a crosslinking agent and a non-reactive liquid additive in the stated mass ratio to obtain a mixture, and adding an initiator to the mixture to obtain a Big Cut resin; Optionally, a low-refractive-index resin monomer may be added to the mixture along with the initiator.

5. The preparation method according to claim 4, characterized in that, The crosslinking agent accounts for 30%-70% of the total mass of the Big Cut resin; the non-reactive liquid additive accounts for 30%-70% of the total mass of the Big Cut resin; the initiator is azobisisobutyronitrile, which accounts for 0.03%-0.1% of the total mass of the Big Cut resin; and the low refractive index resin monomer accounts for 0%-30% of the total mass of the Big Cut resin.

6. The preparation method according to claim 4, characterized in that, The preparation method further includes a pretreatment step for the crosslinking agent, which involves filtering the crosslinking agent using a glass chromatography column, inserting a small amount of degreased cotton at the outlet of the chromatography column, adding alkaline alumina powder, and filtering out the polymerization inhibitor in the crosslinking agent.

7. A plastic embedding method suitable for thick cutting of large-volume samples, characterized in that, The plastic embedding method includes the step of embedding a large volume biological tissue sample with the plastic embedding agent according to any one of claims 1-3 or the plastic embedding agent obtained by the preparation method according to any one of claims 4-6.

8. The plastic embedding method as described in claim 7, characterized in that, The plastic embedding method includes the following steps: (1) Pretreatment: Decalcification and deesterification reagents were used to decalcify and deesterify the biological samples respectively. Then, the fixed biological tissue samples were rinsed multiple times with buffer solution, with the buffer solution being changed every 3-4 hours. (2) Gradient dehydration: The pretreated biological tissue samples were placed in 50% ethanol pre-cooled at 4°C overnight, and then placed in 75% ethanol, 95% ethanol and anhydrous ethanol pre-cooled at 4°C for gradient dehydration for 6-8 hours at each gradient. After that, they were placed in anhydrous ethanol overnight to completely dehydrate the biological tissue samples. (3) Gradient permeation: The plastic embedding agent Big Cut resin was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% Big Cut resin pre-cooled at 4°C overnight, and then placed in 75% and 100% Big Cut resin for gradient permeation, each gradient lasting 6-12 hours; finally, the biological tissue samples were placed in 100% Big Cut resin overnight, then placed in 100% Big Cut resin for 24 hours, and then placed in 100% Big Cut resin for 48 hours to allow the Big Cut resin to fully permeate the biological tissue samples; (4) Polymerization and embedding: The permeated biological tissue sample is placed in a mold, and then 100% BigCut resin is slowly added to the mold to fill it. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

9. The plastic embedding method as described in claim 8, characterized in that, The decalcification reagent and the ester removal reagent mentioned in step (1) are both aqueous solutions; the decalcification reagent is a 10% (w / w) EDTA-2Na solution; the ester removal reagent is prepared from 0.05% (w / w) PI, 10% (w / w) CHAPS, 2% (w / w) Triton X-100, 10% (w / w) DMSO, 1% (w / w) glycine, and 1% (w / w) cyclodextrin; the buffer solution is a 0.01M phosphate buffer; the biological tissue sample is rinsed 2-4 times with the buffer solution.

10. The plastic embedding method as described in claim 8, characterized in that, The thermal polymerization in step (4) is carried out in an oven at a temperature of 36-45°C for a time of 36-72 hours.

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