A plastic embedding method compatible with multicolor fluorescent and immunofluorescent labeling

Abstract

The application discloses a plastic embedding agent BMA / IBOMA resin, monomers of the resin are composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) in a mass ratio of 6:1 to 7:2. The application further discloses a preparation method of the plastic embedding agent, which comprises the steps of mixing the two resins according to the mass percentage, and then adding a crosslinking agent and an initiator. The application further discloses a plastic embedding method compatible with multi-color fluorescence and immunofluorescence labeling, which adopts the BMA / IBOMA resin to embed a biological tissue sample. The embedding method has low cost, and the BMA and IBOMA are used as hydrophobic resin monomers to effectively maintain the fluorescence signal of the sample. In addition, the IBOMA can improve the cutting performance of the sample after embedding, and meet the 1-micron precision cutting requirement of the fMOST system.

Description

Technical Field

[0001] This invention belongs to the field of plastic embedding technology, specifically, it relates to a plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling. Background Technology

[0002] Obtaining detailed structures of the neural networks in large-volume animal models is crucial for understanding the signal transmission mechanisms of the human brain's neural networks and for the diagnosis and treatment of brain diseases. The combination of fluorescent labeling techniques and large-volume fluorescence microscopy can visualize the structure of biological samples, which is of great significance for studying biological tissue structure and exploring disease mechanisms.

[0003] Resin embedding is a well-developed method widely used as a fundamental tool in electron and optical microscopy. For example, embedding fluorescently labeled or stained biological samples in resin followed by imaging with optical imaging systems such as SIM-fMOST can acquire continuous fluorescence signals, reconstructing neuronal projection patterns and fine three-dimensional morphology across the entire brain. Plastic-embedded samples typically undergo perfusion followed by fixation, rinsing, dehydration, infiltration, and polymerization, using organic reagents such as paraformaldehyde, ethanol, and resins. For large-volume fluorescently labeled biological samples, the complex embedding process and the large number of organic reagents involved can lead to quenching of some fluorescent molecules and loss of fine structure after embedding. Therefore, preserving the fluorescent labeling signal and fine structure of biological samples is crucial for plastic embedding.

[0004] Existing plastic embedding methods suffer from low fluorescence retention and poor preservation of fine structure during embedding. Previous methods have addressed this by adjusting resin pH, adding fluorescent protectants during embedding, and lowering polymerization temperature. However, these optimizations are largely based on existing embedding procedures, necessitating a novel embedding method to address these issues. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a novel plastic embedding agent and its application in embedding fluorescently labeled biological tissue samples, effectively preserving the fluorescence signal of biological tissue samples and providing assistance in understanding the signal transmission mechanism of the human brain's neural network and the diagnosis and treatment of brain diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a plastic embedding agent, wherein the embedding agent is BMA / IBOMA resin.

[0008] In one or more embodiments, the monomer of the resin is composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) in a mass ratio of 6:1 to 7:2.

[0009] In a second aspect, the present invention provides a method for preparing the plastic embedding agent, the method comprising the steps of mixing butyl methacrylate and isobornyl methacrylate in the above mass ratio to obtain BMA / IBOMA resin monomer, and adding a crosslinking agent and an initiator to the resin monomer to obtain BMA / IBOMA resin.

[0010] In one or more embodiments, the preparation steps of the BMA / IBOMA resin monomer are as follows: butyl methacrylate and isobornyl methacrylate resin monomers are filtered separately using a glass chromatography column, a small amount of degreased cotton is inserted at the outlet of the chromatography column, alkaline alumina powder is added, the polymerization inhibitor in the resin is filtered out, and then the resin monomers butyl methacrylate and isobornyl methacrylate are mixed in the specified ratio.

[0011] In one or more embodiments, the crosslinking agent is 1,4-butanediol dimethacrylate, which accounts for 10-20% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent and initiator.

[0012] In one or more embodiments, the initiator is azobisisoheptanenitrile, which accounts for 0.1%-0.2% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent and initiator.

[0013] Thirdly, the present invention provides the application of the above-described plastic embedding agent or the plastic embedding agent prepared by any of the above methods in the plastic embedding of biological tissue samples, wherein the biological tissue samples have fluorescent labels or can emit fluorescence.

[0014] Fourthly, the present invention provides a plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling, the plastic embedding method comprising the step of embedding a biological tissue sample having multicolor fluorescence and / or immunofluorescence labeling with the plastic embedding agent described above or the plastic embedding agent obtained by any of the above preparation methods.

[0015] In one or more embodiments, the plastic embedding method includes the following steps:

[0016] (1) Pretreatment: The biological tissue samples were fixed in a fixative at 4°C for 24-48 hours, and then the fixed biological tissue samples were rinsed multiple times with buffer solution, with the buffer solution being changed every 3-4 hours.

[0017] (2) Gradient dehydration: The pretreated biological tissue sample was 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 for 1-2 hours, and then placed in anhydrous ethanol overnight to completely dehydrate the biological tissue sample.

[0018] (3) Gradient permeation: The plastic embedding agent BMA / IBOMA resin was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% BMA / IBOMA resin pre-cooled at 4°C overnight, and then placed in 75% and 100% BMA / IBOMA resin for gradient permeation, each gradient lasting 1-2 hours; finally, the biological tissue samples were placed in 100% BMA / IBOMA resin overnight to allow the BMA / IBOMA resin to fully permeate into the biological tissue samples.

[0019] (4) Polymerization and embedding: The permeation-treated biological tissue sample is placed in a gelatin capsule, and then 100% BMA / IBOMA resin is slowly added to the capsule to fill it. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

[0020] In one or more embodiments, the fixative in step (1) is 4% paraformaldehyde; the buffer is 0.01M phosphate buffer; and the biological tissue sample is rinsed three times with the buffer.

[0021] In one or more embodiments, the thermal polymerization is carried out in an oven at a temperature of 38-45°C for a time of 6-24 hours.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) Compared with existing hydrophobic resins HM20 plastic embedding: The present invention uses BMA / IBOMA resin for plastic embedding, which has a lower cost.

[0024] (2) Using BMA and IBOMA as hydrophobic resin monomers can effectively maintain the fluorescence signal of the sample, and IBOMA can improve the cutting performance of the embedded sample, meeting the 1μm precision cutting requirements of the fMOST system. Attached Figure Description

[0025] Figure 1 This is a flowchart of the preparation method of the embedding agent BMA / IBOMA.

[0026] Figure 2 This is a flowchart of the plastic embedding process of mouse tissues using the embedding agent BMA / IBOMA.

[0027] Figure 3 It is the embedding agent BMA / IBOMA and The plastic embedding fluorescence of HM20 is maintained in contrast.

[0028] Figure 4 This is a fine imaging of the Thy1-GFP mouse brain encapsulated in the embedding agent BMA / IBOMA on fMOST.

[0029] Figure 5 This is a fine imaging of mouse brain lba1 encapsulated in BMA / IBOMA and stained with immunofluorescence on fMOST. Detailed Implementation

[0030] This application establishes a large-sample resin embedding formulation and process method that can meet the requirements of maintaining the fluorescence and immunofluorescence signals of green and red fluorescent proteins after resin embedding and precise cutting of fMOST resin with a thickness of 1 micrometer.

[0031] This application targets and screens hydrophobic resins—specifically BMA resin—to retain the fluorescence signal of samples. Addressing the precision cutting requirements of imaging systems, it introduces IBOMA resin monomers to improve the resin's cutting performance. The effectiveness of BMA / IBOMA resin in retaining sample fluorescence signals was verified against commonly used fluorescent proteins such as EGFP and tdTomato. This was compared with commonly used hydrophobic resins... The embedding methods of HM20 were compared and applied to Thy1-GFP mice and Gad2-cre*Ai14 mice. The results showed that BMA / IBOMA resin can effectively maintain the fluorescence signal and fine structure of the samples.

[0032] The embedding agent BMA / IBOMA resin of the present invention is composed of butyl methacrylate (BMA) and butyl methacrylate (IBOMA) in a mass ratio of 6:1 to 7:2, such as 6:1, 7:1, 7:2, etc.

[0033] In this invention, the BMA resin, namely butyl methacrylate, belongs to the methacrylate class and is commonly used in the production of various polymer materials. It can be copolymerized with other monomers to synthesize copolymers with customized properties. The resulting copolymers can be used in many fields, including automotive coatings, textiles, adhesives, contact lenses, and dental resins. To preserve the BMA resin for a long time, a small amount of polymerization inhibitor is added; therefore, in the preparation of the embedding agent of this invention, the polymerization inhibitor of the BMA resin needs to be filtered out first.

[0034] In this invention, the IBOMA resin, namely isobornyl methacrylate, can be used in heat-resistant plastic optical fibers, adhesives, lithographic ink carriers, modified powder coatings, cleaning coatings, and specialty plastics. It can also be used as a reactive diluent, a comonomer that imparts flexibility, and an agent that improves pigment dispersibility in copolymers. To prevent polymerization, IBOMA resin generally contains polymerization inhibitors, such as trace amounts of hydroquinone. Therefore, in the preparation of the encapsulating agent of this invention, the polymerization inhibitors in the IBOMA resin need to be filtered out first.

[0035] This invention provides a method for preparing the plastic embedding agent, such as... Figure 1 As shown, the method includes the steps of mixing butyl methacrylate and isobornyl methacrylate in the above mass ratio to obtain BMA / IBOMA resin monomers, and adding a crosslinking agent and an initiator to the resin monomers to obtain BMA / IBOMA resin. The final BMA / IBOMA resin structure is as follows. Figure 1 As shown on the right.

[0036] In one or more embodiments, the preparation steps of the BMA / IBOMA resin monomer are as follows: butyl methacrylate and isobornyl methacrylate resin monomers are filtered separately using a glass chromatography column, a small amount of degreased cotton is inserted at the outlet of the chromatography column, alkaline alumina powder is added, the polymerization inhibitor in the resin is filtered out, and then the resin monomers butyl methacrylate and isobornyl methacrylate are mixed in the specified ratio.

[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 BMA / IBOMA 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 1,4-butanediol dimethacrylate, which accounts for 10-20% of the total mass of BMA / IBOMA resin monomer, crosslinking agent and initiator, such as 10%, 15%, 20%, etc.

[0039] 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 BMA / IBOMA resin described in this invention, such as azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (AIHH).

[0040] In one or more embodiments, the initiator is azobisisoheptanenitrile, which accounts for 0.1%-0.2% of the total mass of BMA / IBOMA resin monomer, crosslinking agent and initiator, such as 0.1%, 0.15%, 0.2% etc.

[0041] The BMA / IBOMA resin plastic embedding agent described in this invention can be used for plastic embedding of biological tissue samples, wherein the biological tissue samples are fluorescently labeled or can emit fluorescence.

[0042] It should be understood that, in this field, fluorescent protein and fluorescent dye labeling techniques, combined with whole sample preparation and three-dimensional continuous optical imaging, can visualize the fine structure of large-volume biological tissues, which is of great significance for resolving complex structures in biological tissues. By fully fusing pretreated fluorescently labeled biological tissue with a resin polymer and polymerizing it under certain conditions, the polymerized sample exhibits high rigidity, enabling ultrathin sections of biological tissue for better application in optical imaging. Through continuous research and exploration, the inventors of this invention have discovered that BMA / IBOMA resin can effectively maintain the fluorescence signal of fluorescently labeled biological tissue samples after plastic embedding. The structure of the BMA / IBOMA resin is as follows... Figure 1 As shown on the right. Therefore, BMA / IBOMA resin can be widely used in this field and is of great significance for studying biological tissue structure and exploring disease mechanisms.

[0043] The present invention also includes a plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling, the plastic embedding method comprising the step of embedding a biological tissue sample having multicolor fluorescence and / or immunofluorescence labeling with the plastic embedding agent described above or a plastic embedding agent obtained by any of the preparation methods described above.

[0044] In one or more embodiments, the plastic embedding method includes the following steps:

[0045] (1) Pretreatment: The biological tissue samples were fixed in a fixative at 4°C for 24-48 hours, and then the fixed biological tissue samples were rinsed multiple times with buffer solution, with the buffer solution being changed every 3-4 hours.

[0046] (2) Gradient dehydration: The pretreated biological tissue sample was 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 for 1-2 hours, and then placed in anhydrous ethanol overnight to completely dehydrate the biological tissue sample.

[0047] (3) Gradient permeation: The plastic embedding agent BMA / IBOMA resin was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% BMA / IBOMA resin pre-cooled at 4°C overnight, and then placed in 75% and 100% BMA / IBOMA resin for gradient permeation, with each gradient lasting 1-2 hours; finally, the biological tissue samples were placed in 100% BMA / IBOMA resin overnight to allow the BMA / IBOMA resin to fully permeate the biological tissue samples.

[0048] (4) Polymerization and embedding: The permeated biological tissue sample is placed in a gelatin capsule, and then 100% BMA / IBOMA resin is slowly added to the capsule to fill it. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

[0049] In one or more embodiments, the fixative in step (1) is 4% paraformaldehyde; the buffer is 0.01M phosphate buffer; and the biological tissue sample is rinsed three times with the buffer.

[0050] In one or more embodiments, the thermal polymerization is carried out in an oven at a temperature of 38-45°C for a time of 6-24 hours.

[0051] 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 fluorescently labeled biological tissue samples are embedded in the embedding agent described in this invention, which can effectively preserve the fluorescence signal and fine structure of the samples.

[0052] It should be understood that, in order to verify the embedding effect of BMA / IBOMA resin, animal tissues can be used for experiments, such as using fluorescently labeled mouse tissues to verify the fluorescence expression after embedding and sectioning.

[0053] As an example, such as Figure 2The diagram illustrates the steps involved in embedding mouse tissue using BMA / IBOMA resin. Of course, for different biological tissue samples, minor adjustments to steps or reagents may be required during the embedding process; these adjustments are standard procedure for those skilled in the art.

[0054] 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) is completed, the animal tissue can be acidified. For example, the animal tissue can be placed in 100% BMA / IBOMA resin containing 25μl / 10ml acetic acid for 12-14 hours to acidify the animal tissue; and then subsequent steps can be carried out.

[0055] Example

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] In the following examples and comparative examples, the BMA resin was purchased from Sigma; the IBOMA resin was purchased from Bidex Pharmaceuticals; the crosslinking agent 1,4-butanediol dimethacrylate was purchased from aladdin; the initiator azobisisoheptanenitrile was purchased from Adamas; and the acrylate monomers used to manufacture the HM20 resin were purchased from Sigma.

[0062] Example 1: A method for preparing a plastic embedding agent BMA / IBOMA resin

[0063] A plastic embedding agent BMA / IBOMA resin, wherein the monomers of the resin are composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) in a mass ratio of 7:2.

[0064] The preparation steps of the above-mentioned BMA / IBOMA resin are as follows: butyl methacrylate and isobornyl methacrylate resin monomers are filtered separately using a glass chromatography column. A small amount of degreased cotton is inserted at the outlet of the chromatography column, and alkaline alumina powder is added to filter out the polymerization inhibitor in the resin. The resin monomers butyl methacrylate and isobornyl methacrylate are then mixed in the specified ratio to obtain the BMA / IBOMA resin monomer. A crosslinking agent and an initiator are then added to the monomer to react and obtain the BMA / IBOMA resin. The crosslinking agent is 1,4-butanediol dimethacrylate, which accounts for 10% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent, and initiator. The initiator, azobisisobutyronitrile, accounts for 0.2% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent, and initiator.

[0065] Example 2: A method for preparing a plastic embedding agent BMA / IBOMA resin

[0066] A plastic embedding agent BMA / IBOMA resin, wherein the monomers of the resin are composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) in a mass ratio of 13:3.

[0067] The preparation steps of the above-mentioned BMA / IBOMA resin are as follows: butyl methacrylate and isobornyl methacrylate resin monomers are filtered separately using a glass chromatography column. A small amount of degreased cotton is inserted at the outlet of the chromatography column, and alkaline alumina powder is added to filter out the polymerization inhibitor in the resin. The resin monomers butyl methacrylate and isobornyl methacrylate are then mixed in the specified ratio to obtain the BMA / IBOMA resin monomer. A crosslinking agent and an initiator are then added to the monomer to react and obtain the BMA / IBOMA resin. The crosslinking agent is 1,4-butanediol dimethacrylate, which accounts for 15% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent, and initiator. The initiator, azobisisobutyronitrile, accounts for 0.15% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent, and initiator.

[0068] Example 3: A method for preparing a plastic embedding agent BMA / IBOMA resin

[0069] A plastic embedding agent BMA / IBOMA resin, wherein the monomers of the resin are composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) in a mass ratio of 6:1.

[0070] The preparation steps of the above-mentioned BMA / IBOMA resin are as follows: butyl methacrylate and isobornyl methacrylate resin monomers are filtered separately using a glass chromatography column. A small amount of degreased cotton is inserted at the outlet of the chromatography column, and alkaline alumina powder is added to filter out the polymerization inhibitor in the resin. The resin monomers butyl methacrylate and isobornyl methacrylate are then mixed in the specified ratio to obtain the BMA / IBOMA resin monomer. A crosslinking agent and an initiator are then added to the monomer to react and obtain the BMA / IBOMA resin. The crosslinking agent is 1,4-butanediol dimethacrylate, which accounts for 20% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent, and initiator. The initiator, azobisisobutyronitrile, accounts for 0.1% of the total mass of the BMA / IBOMA resin monomer, crosslinking agent, and initiator.

[0071] Comparative Example 1: A plastic embedding agent Preparation method of HM20 resin

[0072] A plastic embedding agent HM20 resin, said resin containing 82.5% by mass of acrylate monomers.

[0073] The preparation steps of the above HM20 resin are as follows: 82.5% of acrylate monomers, 17.3% of crosslinking agent triethylene glycol dimethacrylate and 0.2% of initiator azobisisobutyronitrile are mixed and dissolved according to the mass percentage.

[0074] Application Example 1: Comparative Test of Fluorescence Retention between BMA / IBOMA Resin and HM20 Plastic Embedding

[0075] Thy1-GFP and Gad2-cre*Ai14 mice (expressing EGFP and Tdtomato fluorescent proteins, respectively) were anesthetized with chloral hydrate, and perfused and fixed via cardiac perfusion. The mice were then perfused and fixed with 0.01 M phosphate buffer and 4% paraformaldehyde, and their brains were removed.

[0076] Mouse brains were placed in 4% paraformaldehyde and placed in an environment at 4°C for 24 hours for post-fixation.

[0077] The mouse brains were then rinsed with 0.01M phosphate buffer, with the buffer changed every 3-4 hours, for a total of 3 rinses.

[0078] After rinsing, the rinsed mouse brain was cut into 50μm thick slices using a vibrating slicer.

[0079] Select several adjacent brain slices, place them flat on a glass slide, cover with a coverslip, use confocal microscopy on the same cortical region, ensuring that the imaging parameters remain consistent, and record the imaging position.

[0080] After imaging, the samples were placed in 50%, 75%, and 95% ethanol for gradient dehydration, with each gradient lasting 5 minutes.

[0081] Soak the brain slices in anhydrous ethanol for 10 minutes, repeating twice, to ensure complete dehydration.

[0082] The BMA / IBOMA resin prepared in Example 1 and the HM20 resin prepared in Comparative Example 1 were diluted with anhydrous ethanol to prepare BMA / IBOMA resin and HM20 resin with a mass ratio of 50% and 75% respectively.

[0083] After complete dehydration, the brain slices were placed in 50% and 75% BMA / IBOMA resin and HM20 resin for gradient permeation, with each gradient lasting 10 minutes.

[0084] Immerse the brain slices in 100% BMA / IBOMA resin and HM20 resin for 15 minutes, repeating twice to ensure thorough penetration.

[0085] Transfer the brain slice to a coverslip, add BMA / IBOMA resin and HM20 resin to the brain slice, and then cover it with a coverslip.

[0086] The polymerization was carried out in an oven at a temperature of 45°C for 6 hours.

[0087] Use confocal imaging to image the same area of ​​the embedded brain slice, ensuring that the imaging parameters remain consistent.

[0088] Confocal images of brain slices taken before and after embedding were analyzed. The average fluorescence intensity of cell bodies was analyzed using ImageJ software, and the fluorescence retention rate of the brain slices after resin embedding was calculated.

[0089] The results of the brain slice comparison were referenced. Figure 3 The results show that, compared with HM20 resin embedding, biological tissue samples embedded with BMA / IBOMA resin have a higher fluorescence retention rate.

[0090] Application Example 2: BMA / IBOMA Resin Embedded fMOST Imaging Test

[0091] Thy1-GFP mice were anesthetized with chloral hydrate, and perfusion and fixation were performed using a cardiac perfusion method. Mice were perfused and fixed using 0.01M phosphate buffer and 4% paraformaldehyde, and the brains were removed.

[0092] Mouse brains were placed in 4% paraformaldehyde and left at 4°C for 24-48 hours for post-fixation.

[0093] The mouse brains were then rinsed with 0.01M phosphate buffer, with the buffer changed every 3-4 hours, for a total of 3 rinses.

[0094] After rinsing, the rinsed mouse brains were placed in 50% ethanol pre-cooled at 4°C overnight.

[0095] The mixture was subjected to gradient dehydration in 75% ethanol, 95% ethanol and anhydrous ethanol for 1-2 hours per gradient.

[0096] The rat brains were placed in anhydrous ethanol overnight to completely dehydrate them.

[0097] The BMA / IBOMA resin prepared in Example 2 was diluted with anhydrous ethanol to prepare BMA / IBOMA resins with a mass ratio of 50% and 75%, respectively, for later use.

[0098] After complete dehydration, the mouse brains were placed in 50% BMA / IBOMA resin pre-cooled at 4°C overnight.

[0099] Gradient permeation was performed in 75% and 100% BMA / IBOMA resins for 1-2 hours per gradient.

[0100] Soak overnight in 100% BMA / IBOMA resin.

[0101] The mice were immersed in 100% BMA / IBOMA resin for one day to allow for full penetration of the brain.

[0102] The rat brains were acidified by immersing them in 100% BMA / IBOMA resin containing 25 μl / 10 ml acetic acid for 12-14 hours.

[0103] The transferred and permeated mouse brain was placed in a 000 gelatin capsule, and then 100% BMA / IBOMA resin was slowly added into the capsule for encapsulation.

[0104] The polymerization was carried out in an oven at a temperature of 38°C for 12 hours.

[0105] The embedded mouse brain was imaged using the fMOST system with 1μm precision ablation in 0.6M alkaline buffer. The resulting mouse brain fluorescence images were compared with [previous data]. Figure 4 .according to Figure 4It can be seen that mouse brains embedded in BMA / IBOMA resin exhibit high fluorescence retention on the fMOST system, and the fluorescence signal used to label neurons is bright. Figure 4 (Left); It maintains the fine structure of neurons, which can meet the requirements of precision cutting. Figure 4 (Right, 100 layers of 3D image cut with 1μm).

[0106] Application Example 3: Fine imaging of BMA / IBOMA-embedded mouse brain LBA1 immunofluorescence staining on fMOST.

[0107] Mouse brains were stained with LBA1 immunofluorescence and incubated overnight in 50% ethanol pre-cooled at 4°C.

[0108] The mixture was subjected to gradient dehydration in 75% ethanol, 95% ethanol and anhydrous ethanol for 1-2 hours per gradient.

[0109] The rat brains were placed in anhydrous ethanol overnight to completely dehydrate them.

[0110] The BMA / IBOMA resin prepared in Example 3 was diluted with anhydrous ethanol to prepare BMA / IBOMA resins with a mass ratio of 50% and 75%, respectively, for later use.

[0111] After complete dehydration, the mouse brains were placed in 50% BMA / IBOMA resin pre-cooled at 4°C overnight.

[0112] Gradient permeation was performed in 75% and 100% BMA / IBOMA resins, with each gradient lasting 1-2 hours.

[0113] Soak overnight in 100% BMA / IBOMA resin.

[0114] The mice were immersed in 100% BMA / IBOMA resin for one day to allow for full penetration of the brain.

[0115] The rat brains were acidified by immersing them in 100% BMA / IBOMA resin containing 25 μl / 10 ml acetic acid for 12-14 hours.

[0116] The transferred and permeated mouse brain was placed in a 000 gelatin capsule, and then 100% BMA / IBOMA resin was slowly added into the capsule for encapsulation.

[0117] The polymerization was carried out in an oven at a temperature of 45°C for 12 hours.

[0118] The embedded mouse brain was imaged using the fMOST system with 1μm precision cutting in pure water. The resulting immunofluorescence images of mouse brain microglia are shown below. Figure 5As shown, the BMA / IBOMA resin described in this invention can effectively preserve the fluorescence signal of biological tissue samples after embedding them.

[0119] The above description is merely an embodiment of the present invention. It should be noted that those skilled in the art can make changes without departing from the concept of the present invention, and these changes all fall within the protection scope of the present invention.

Claims

1. The application of a plastic embedding agent in the shaping and embedding of biological tissue samples, characterized in that, The embedding agent is BMA / IBOMA resin, wherein the monomers of the resin are composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) in a mass ratio of 6:1 to 7:2; the biological tissue sample is fluorescently labeled or can emit fluorescence.

2. The application as described in claim 1, characterized in that, The plastic embedding agent used in the application is prepared by a method comprising the steps of mixing butyl methacrylate and isobornyl methacrylate in the mass ratio to obtain BMA / IBOMA resin monomer, and adding a crosslinking agent and an initiator to the resin monomer to obtain BMA / IBOMA resin.

3. The application as described in claim 2, characterized in that, The preparation steps of the BMA / IBOMA resin monomer are as follows: butyl methacrylate and isobornyl methacrylate resin monomers are filtered separately using a glass chromatography column. A small amount of degreased cotton is inserted at the outlet of the chromatography column, and alkaline alumina powder is added to filter out the polymerization inhibitor in the resin. Then, the resin monomers butyl methacrylate and isobornyl methacrylate are mixed in the specified ratio.

4. The application as described in claim 2, characterized in that, The crosslinking agent is 1,4-butanediol dimethacrylate, which accounts for 10-20% of the total mass of BMA / IBOMA resin monomer, crosslinking agent and initiator.

5. The application as described in claim 2, characterized in that, The initiator is azobisisoheptanenitrile, which accounts for 0.1%-0.2% of the total mass of BMA / IBOMA resin monomer, crosslinking agent and initiator.

6. A plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling, characterized in that, The plastic embedding method includes the step of embedding a biological tissue sample labeled with multicolor fluorescence and / or immunofluorescence using the plastic embedding agent used in any one of claims 1-5.

7. The plastic embedding method as described in claim 6, characterized in that, The plastic embedding method includes the following steps: (1) Pretreatment: biological tissue samples were fixed in a fixative at 4°C for 24-48 hours, and 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, each gradient lasting 1-2 hours. After that, the samples were placed in anhydrous ethanol overnight to completely dehydrate the biological tissue samples. (3) Gradient permeation: The plastic embedding agent BMA / IBOMA resin was diluted to 50% and 75% concentrations with anhydrous ethanol; the completely dehydrated biological tissue samples were placed in 50% BMA / IBOMA resin pre-cooled at 4°C overnight, and then placed in 75% and 100% BMA / IBOMA resin for gradient permeation, each gradient lasting 1-2 hours; finally, the biological tissue samples were placed in 100% BMA / IBOMA resin overnight, and then placed in 100% BMA / IBOMA resin for one day to allow the BMA / IBOMA resin to fully permeate the biological tissue samples. (4) Polymerization and embedding: The permeated biological tissue sample is placed in a gelatin capsule, and then 100% BMA / IBOMA resin is slowly added into the capsule to fill it. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

8. The plastic embedding method as described in claim 7, characterized in that, The fixative in step (1) is 4% paraformaldehyde; the buffer solution is 0.01M phosphate buffer; and the biological tissue sample is rinsed three times with the buffer solution.

9. The plastic embedding method as described in claim 7, characterized in that, The thermal polymerization is carried out in an oven at a temperature of 38-45°C for 6-24 hours.

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