Plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling

Abstract

The invention discloses a plastic embedding medium BMA / IBOMA resin. A monomer of the resin is composed of butyl methacrylate (BMA) and isobornyl methacrylate (IBOMA) according to a mass ratio of 6: 1-7: 2. The invention also discloses a preparation method of the plastic embedding agent, which comprises the following steps: mixing the two resins according to the mass percentage, and then adding the cross-linking agent and the initiator. The invention further discloses a plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling. According to the plastic embedding method, a biological tissue sample is embedded by adopting the BMA / IBOMA resin. The embedding method is low in cost, BMA and IBOMA are used as hydrophobic resin monomers, fluorescence signals of the sample can be effectively kept, in addition, IBOMA can improve the cutting performance of the embedded sample, and the 1-micron precision cutting requirement of an fMOST system is met.

Description

Technical Field

[0001] The invention belongs to the technical field of plastic embedding, and in particular, relates to a plastic embedding method compatible with multi-color fluorescence and immunofluorescence labeling. Background Art

[0002] Obtaining the fine structure of the brain neural network of large-volume model animals is helpful for understanding the signal transmission mechanism of the human brain neural network and the diagnosis and treatment of brain diseases. The combination of fluorescent labeling technology and large-volume fluorescence microscopy technology can visualize the structure of biological samples, which is of great significance for studying the structure of biological tissues and exploring the mechanism of diseases.

[0003] Resin embedding is a well-developed method that is widely used as a basic tool for electron microscopy and optical microscopy. For example, biological samples labeled with fluorescent proteins or stained with fluorescent dyes can be embedded in resin and then sliced ​​and imaged with an optical imaging system such as SIM-fMOST to obtain continuous fluorescence signals and reconstruct the neuronal projection connection pattern and three-dimensional fine morphology of neurons in the whole brain. Plastic-embedded samples usually undergo post-perfusion fixation, rinsing, dehydration, infiltration, and polymerization. This process uses organic reagents such as paraformaldehyde, ethanol, and resin. For large-volume fluorescent-labeled biological samples, the embedding process is complicated and involves more organic reagents, which leads to the quenching of some fluorescent molecules in the sample after embedding and the loss of fine structure. Therefore, maintaining the fluorescent labeling signal and fine structure of biological samples is the key to plastic embedding.

[0004] The problem with the existing plastic embedding method is that the fluorescence retention rate of the sample during the embedding process is low, and the fine structure retention effect is poor. In the past, plastic embedding was solved by adjusting the pH of the resin, adding a fluorescent protective agent during the resin embedding process, and lowering the polymerization temperature. However, the above optimization methods are basically based on the optimization of the existing embedding process, so a new embedding method is needed to solve the above problems. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a new plastic embedding agent and its application in embedding fluorescently labeled biological tissue samples, so as to effectively maintain the fluorescent signal of the biological tissue samples, and provide assistance for the signal transmission mechanism of the human brain neural network and the diagnosis and treatment of brain diseases.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

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

[0008] In one or more embodiments, 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.

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

[0010] In one or more embodiments, the preparation steps of the BMA / IBOMA resin monomer are: using a glass chromatography column to filter butyl methacrylate and isobornyl methacrylate resin monomers respectively, plugging some absorbent cotton at the outlet of the chromatography column, adding basic alumina powder, filtering out the inhibitor in the resin, and then mixing the resin monomers butyl methacrylate and isobornyl methacrylate according to the said proportion.

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

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

[0013] In a third aspect, the present invention provides the use of the above-mentioned plastic embedding agent or the plastic embedding agent obtained by any of the preparation methods described above in the plastic embedding of biological tissue samples, wherein the biological tissue samples have fluorescent markers or can emit fluorescence.

[0014] In a fourth aspect, the present invention provides a plastic embedding method that is compatible with multi-color fluorescence and immunofluorescence labeling, the plastic embedding method comprising the step of embedding a biological tissue sample having multi-color fluorescence and / or immunofluorescence labeling with the above-mentioned plastic embedding agent or a plastic embedding agent obtained by any of the preparation methods described above.

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

[0016] (1) Pretreatment: Fix the biological tissue sample with a fixative at 4°C for 24-48 hours, then rinse the fixed biological tissue sample with a buffer solution for multiple times, replacing the buffer solution every 3-4 hours;

[0017] (2) Gradient dehydration: The pretreated biological tissue sample is placed in 50% ethanol precooled at 4°C overnight, and then placed in 75%, 95% ethanol and anhydrous ethanol precooled 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 infiltration: dilute the plastic embedding agent BMA / IBOMA resin to 50% and 75% concentrations with anhydrous ethanol; place the completely dehydrated biological tissue sample in 50% BMA / IBOMA resin pre-cooled at 4° C. overnight, and then place it in 75% and 100% BMA / IBOMA resin for gradient infiltration, each gradient for 1-2 hours; finally, place the biological tissue sample in 100% BMA / IBOMA resin overnight to allow the BMA / IBOMA resin to fully penetrate into the biological tissue sample;

[0019] (4) Polymerization embedding: The biological tissue sample after infiltration treatment is placed in a gelatin capsule, and then 100% BMA / IBOMA resin is slowly added into the capsule to fill the capsule, and finally thermal polymerization is performed to complete the embedding of the biological tissue sample.

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

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

[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 cost of plastic embedding using BMA / IBOMA resin is low;

[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 sample after embedding, meeting the 1μm precision cutting requirements of the fMOST system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a flow chart of the preparation method of embedding agent BMA / IBOMA.

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

[0027] Figure 3 Is the embedding agent BMA / IBOMA and The plastic-embedded fluorescence of HM20 maintains contrast.

[0028] Figure 4 The Thy1-GFP mouse brain embedded in BMA / IBOMA was imaged in detail on fMOST.

[0029] Figure 5 The embedding medium BMA / IBOMA was used to embed the mouse brain lba1 and the immunofluorescence staining was finely imaged on fMOST. DETAILED DESCRIPTION

[0030] This application establishes a large sample resin embedding formula and process method, which can meet the requirements of maintaining green and red fluorescent protein fluorescence signals and immunofluorescence signals after resin embedding and precise cutting of 1 micron thick fMOST resin.

[0031] This application targeted the screening of hydrophobic resins - BMA resins to maintain the fluorescence signal of the sample. In view of the need for precision cutting of the imaging system, the IBOMA resin monomer was introduced to improve the cutting performance of the resin. The effectiveness of BMA / IBOMA resin in maintaining the fluorescence signal of the sample was verified on commonly used fluorescent proteins such as EGFP and tdTomato. The embedding methods of HM20 were compared and applied to Thy1-GFP mouse and Gad2-cre*Ai14 mouse samples. The results showed that BMA / IBOMA resin can effectively maintain the fluorescent signal and fine structure of the samples.

[0032] The embedding agent BMA / IBOMA resin of the present invention has monomers 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 the present invention, the BMA resin, i.e. butyl methacrylate, belongs to the class of methacrylates 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. In order to preserve the BMA resin for a long time, a small amount of polymerization inhibitor is added thereto, so the polymerization inhibitor of the BMA resin needs to be filtered out first in the preparation of the embedding agent of the present invention.

[0034] In the present invention, the IBOMA resin, i.e., isobornyl methacrylate, can be used in the fields of heat-resistant plastic optical fibers, adhesives, lithographic ink vehicles, modified powder coatings, clean coatings, and special plastics, and can also be used as a reactive diluent, as a comonomer that imparts flexibility, and as a pigment dispersant that can improve the copolymer. In order to prevent polymerization, IBOMA resins are generally added with polymerization inhibitors, such as a trace amount of hydroquinone, etc., so the polymerization inhibitor of the IBOMA resin needs to be filtered out in the preparation of the embedding agent of the present invention.

[0035] The present 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 monomer, and adding a crosslinking agent and an initiator to the resin monomer to obtain BMA / IBOMA resin. The structure of the finally obtained BMA / IBOMA resin is as follows: Figure 1 Shown on the right.

[0036] In one or more embodiments, the preparation steps of the BMA / IBOMA resin monomer are: use a glass chromatography column to filter butyl methacrylate and isobornyl methacrylate resin monomers respectively, plug some cotton wool at the outlet of the chromatography column, add basic alumina powder, filter out the inhibitor in the resin, and then mix the resin monomers butyl methacrylate and isobornyl methacrylate according to the said proportion.

[0037] It should be understood that the crosslinking agent described in the present invention is common knowledge in the art, also called curing agent, hardener, curing agent, etc. It can transform linear or slightly branched macromolecules into a three-dimensional network structure, thereby improving strength, heat resistance, wear resistance, solvent resistance, etc. In the art, any chemical substance with the same effect can be tried in the preparation of the BMA / IBOMA resin described in the present invention, such as triethylene glycol dimethacrylate, 1,4-butylene glycol dimethacrylate, etc.

[0038] In one or more embodiments, the crosslinking agent is 1,4-butylene glycol dimethacrylate, which accounts for 10-20% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator, such as 10%, 15%, 20%, etc.

[0039] It should be understood that the initiator described in the present invention is common knowledge in the art. An initiator is a substance that can initiate a monomer to undergo a polymerization reaction. The polymerization active centers of unsaturated monomers include free radical type, anionic type, cationic type, and coordination compound, etc. The most widely used in the adhesive industry is the free radical type, which exhibits unique chemical activity. Under the action of heat or light, the covalent bond is homolytically cleaved to generate two free radicals, which can initiate a polymerization reaction. In the art, any chemical substance with the same effect can be attempted to be used in the preparation of the BMA / IBOMA resin described in the present invention, such as azobisisobutyronitrile, azobisisoheptanenitrile, etc.

[0040] 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, crosslinker and initiator, such as 0.1%, 0.15%, 0.2%, etc.

[0041] The plastic embedding agent BMA / IBOMA resin of the present invention can be used for plastic embedding of biological tissue samples, wherein the biological tissue samples have fluorescent markers or can emit fluorescence.

[0042] It should be understood that in this field, fluorescent protein and fluorescent dye labeling technology combined with complete sample preparation and three-dimensional continuous optical imaging can visualize the fine structure of large-volume biological tissues, which is of great significance for analyzing the complex structures in biological tissues. The biological tissue with fluorescent labels after pretreatment is fully fused with the resin polymer and polymerized under certain conditions. The polymerized sample has a higher hardness, which can achieve ultra-thin sections of biological tissues and is better used in optical imaging. After continuous research and exploration, the inventors of the present invention found that BMA / IBOMA resin can effectively maintain the fluorescent signal of the sample after plastic embedding of biological tissue samples with fluorescent labels. The structure of the BMA / IBOMA resin is as follows: Figure 1 As shown on the right, 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 multi-color fluorescence and immunofluorescence labeling, the plastic embedding method comprising the step of embedding a biological tissue sample having multi-color fluorescence and / or immunofluorescence labeling with the above-mentioned plastic embedding agent or a plastic embedding agent obtained by any of the preparation methods described above.

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

[0045] (1) Pretreatment: Fix the biological tissue sample with a fixative at 4°C for 24-48 hours, then rinse the fixed biological tissue sample with a buffer solution for multiple times, replacing the buffer solution every 3-4 hours;

[0046] (2) Gradient dehydration: The pretreated biological tissue sample is placed in 50% ethanol precooled at 4°C overnight, and then placed in 75%, 95% ethanol and anhydrous ethanol precooled 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 infiltration: dilute the plastic embedding agent BMA / IBOMA resin to 50% and 75% concentrations with anhydrous ethanol; place the completely dehydrated biological tissue sample in 50% BMA / IBOMA resin precooled at 4° C. overnight, and then place it in 75% and 100% BMA / IBOMA resin for gradient infiltration, each gradient lasting 1-2 hours; finally, place the biological tissue sample in 100% BMA / IBOMA resin overnight to allow the BMA / IBOMA resin to fully penetrate the biological tissue sample;

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

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

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

[0051] It should be understood that the biological tissue samples described in the present invention can be human tissue or animal tissue, and the specific tissue is not limited, such as tissues in different parts of the body, such as all or part of the brain, heart, trunk, spinal cord tissue, etc. These fluorescently labeled biological tissue samples can be embedded in the embedding agent described in the present invention to effectively maintain the fluorescent signal and fine structure of the sample.

[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 slices.

[0053] As an example, Figure 2The figure shows the process flow of embedding mouse tissue using BMA / IBOMA resin. Of course, for different biological tissue samples, the embedding process may involve subtle steps or reagent adjustments, which are routine operations of those skilled in the art.

[0054] It should be understood that the plastic embedding method described in the present invention is not fixed. Those skilled in the art can make appropriate adjustments to the steps or specific parameters according to different animal tissues to adapt to different studies. For example, the animal tissue can be acidified after step (3). For example, the animal tissue can be placed in 100% BMA / IBOMA resin containing 25 μl / 10 ml acetic acid for 12-14 hours to acidify the animal tissue; and then proceed to subsequent steps.

[0055] Example

[0056] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] Before further describing the 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 terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0058] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0059] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0060] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0061] The BMA resin in the following examples and comparative examples was purchased from Sigma; the IBOMA resin was purchased from Bitartrate Pharmaceuticals; the crosslinking agent 1,4-butanediol dimethacrylate was purchased from Aladdin; the initiator azobisisoheptanenitrile was purchased from Adamas; and the acrylate monomer used to make the HM20 resin was purchased from Sigma.

[0062] Example 1: Preparation method of a plastic embedding agent BMA / IBOMA resin

[0063] A plastic embedding agent BMA / IBOMA resin, wherein 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: using a glass chromatography column to filter butyl methacrylate and isobornyl methacrylate resin monomers respectively, plugging some absorbent cotton at the outlet of the chromatography column, adding alkaline alumina powder, filtering out the inhibitor in the resin, and then mixing the resin monomers butyl methacrylate and isobornyl methacrylate in the proportion to obtain BMA / IBOMA resin monomers, and then adding a crosslinking agent and an initiator to the monomers for reaction to obtain the BMA / IBOMA resin, wherein the crosslinking agent is 1,4-butylene glycol dimethacrylate, which accounts for 10% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator; wherein the initiator is azobisisoheptonitrile, which accounts for 0.2% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator.

[0065] Example 2: Preparation method of a plastic embedding agent BMA / IBOMA resin

[0066] A plastic embedding agent BMA / IBOMA resin, wherein 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: using a glass chromatography column to filter butyl methacrylate and isobornyl methacrylate resin monomers respectively, plugging some absorbent cotton at the outlet of the chromatography column, adding alkaline alumina powder, filtering out the inhibitor in the resin, and then mixing the resin monomers butyl methacrylate and isobornyl methacrylate in the said proportion to obtain BMA / IBOMA resin monomers, and then adding a crosslinking agent and an initiator to the monomers for reaction to obtain the BMA / IBOMA resin, wherein the crosslinking agent is 1,4-butylene glycol dimethacrylate, which accounts for 15% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator; wherein the initiator is azobisisoheptonitrile, which accounts for 0.15% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator.

[0068] Example 3: Preparation method of a plastic embedding agent BMA / IBOMA resin

[0069] A plastic embedding agent BMA / IBOMA resin, wherein 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: using a glass chromatography column to filter butyl methacrylate and isobornyl methacrylate resin monomers respectively, plugging some absorbent cotton at the outlet of the chromatography column, adding alkaline alumina powder, filtering out the inhibitor in the resin, and then mixing the resin monomers butyl methacrylate and isobornyl methacrylate according to the said proportion to obtain BMA / IBOMA resin monomers, and then adding a crosslinking agent and an initiator to the monomers for reaction to obtain the BMA / IBOMA resin, wherein the crosslinking agent is 1,4-butylene glycol dimethacrylate, which accounts for 20% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator; wherein the initiator is azobisisoheptonitrile, which accounts for 0.1% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator.

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

[0072] A plastic embedding agent HM20 resin contains 82.5% by mass of acrylic acid ester monomers.

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

[0074] Application Example 1: Comparative test of fluorescence retention of BMA / IBOMA resin and HM20 plastic embedding

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

[0076] The mouse brain was placed in 4% paraformaldehyde at 4°C for 24 hours for post-fixation.

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

[0078] After rinsing, the rinsed mouse brain was cut into 50 μm thick brain slices on a vibration microtome.

[0079] Select several adjacent brain slices, flatten them on a glass slide, cover them with a coverslip, use confocal microscopy on the same cortical area, keep the imaging parameters consistent, and record the imaging position.

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

[0081] Place in anhydrous ethanol for 10 minutes, repeat twice to completely dehydrate the brain slices.

[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 50% and 75% by mass of BMA / IBOMA resin, and 50% and 75% by mass of HM20 resin.

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

[0084] Place the slices in 100% BMA / IBOMA resin and HM20 resin for 15 minutes, repeat twice to ensure that the slices are fully infiltrated.

[0085] The brain slices were transferred to coverslips, BMA / IBOMA resin and HM20 resin were added dropwise on the brain slices, and then covered with coverslips.

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

[0087] Use confocal microscopy to image the same area of ​​the embedded brain slices, keeping the imaging parameters consistent.

[0088] Confocal images of brain slices taken before and after embedding were analyzed. Image J software was used to analyze the average fluorescence intensity of cell bodies and calculate the fluorescence retention rate of brain slices after resin embedding.

[0089] The obtained brain slice comparison results refer to Figure 3 According to the results, compared with HM20 resin embedding, biological tissue samples embedded in 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, perfused and fixed by cardiac perfusion, perfused and fixed with 0.01M phosphate buffer solution and 4% paraformaldehyde, and the mouse brain was removed.

[0092] The mouse brain was placed in 4% paraformaldehyde at 4°C for 24-48 hours for post-fixation.

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

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

[0095] The sample was placed in 75%, 95% ethanol and anhydrous ethanol for gradient dehydration, with each gradient taking 1-2 hours.

[0096] The mouse brain was placed in anhydrous ethanol overnight to completely dehydrate it.

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

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

[0099] Gradient infiltration was performed in 75% and 100% BMA / IBOMA resins, with each gradient taking 1-2 hours.

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

[0101] The rat brain was placed in 100% BMA / IBOMA resin for one day to allow for full penetration.

[0102] The rat brain was acidified by placing it in 100% BMA / IBOMA resin containing 25 μl / 10 ml acetic acid for 12-14 hours.

[0103] The permeated mouse brain was transferred into 000 gelatin capsules, and then 100% BMA / IBOMA resin was slowly added into the capsules for embedding.

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

[0105] The embedded mouse brain was imaged on the fMOST system and subjected to 1 μm precision cutting in 0.6 M alkaline buffer. The obtained mouse brain fluorescence image was referenced Figure 4 .according to Figure 4It can be seen that the mouse brain embedded with BMA / IBOMA resin has a high fluorescence retention rate when imaged on the fMOST system, and the fluorescence signal used to mark neurons is bright ( Figure 4 Left); The fine structure of neurons is maintained to meet the needs of precision cutting ( Figure 4 Right, 3D image of 100 slices cut at 1 μm).

[0106] Application Example 3: Detailed imaging of lba1 immunofluorescence staining of BMA / IBOMA-embedded mouse brain on fMOST

[0107] Mouse brains were placed in 50% ethanol precooled at 4 °C overnight for immunofluorescence staining of lba1.

[0108] The sample was placed in 75%, 95% ethanol and anhydrous ethanol for gradient dehydration, with each gradient taking 1-2 hours.

[0109] The mouse brain was placed in anhydrous ethanol overnight to completely dehydrate it.

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

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

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

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

[0114] The rat brain was placed in 100% BMA / IBOMA resin for one day to allow for full penetration.

[0115] The rat brain was acidified by placing it in 100% BMA / IBOMA resin containing 25 μl / 10 ml acetic acid for 12-14 hours.

[0116] The permeated mouse brain was transferred into 000 gelatin capsules, and then 100% BMA / IBOMA resin was slowly added into the capsules for embedding.

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

[0118] The embedded mouse brain was imaged on the fMOST system and 1 μm precision cutting was performed in pure water. The obtained immunofluorescence image of microglia in the mouse brain is shown in the following figure. Figure 5As shown, it is shown that after the BMA / IBOMA resin of the present invention is used to embed the biological tissue sample, the fluorescence signal of the sample can be effectively maintained.

[0119] The above is only an implementation method of the present invention. It should be pointed out that, for ordinary technicians in this field, changes can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A plastic embedding agent, characterized in that The embedding agent is BMA / IBOMA resin, and 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.

2. The method for preparing the plastic embedding agent according to claim 1, characterized in that: The method comprises the steps of mixing butyl methacrylate and isobornyl methacrylate in the 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.

3. The preparation method according to claim 2, characterized in that: The preparation steps of the BMA / IBOMA resin monomer are as follows: using a glass chromatography column to filter butyl methacrylate and isobornyl methacrylate resin monomers respectively, plugging some absorbent cotton at the outlet of the chromatography column, adding alkaline alumina powder, filtering out the inhibitor in the resin, and then mixing the resin monomers butyl methacrylate and isobornyl methacrylate according to the ratio.

4. The preparation method according to claim 2, characterized in that: The crosslinking agent is 1,4-butylene glycol dimethacrylate, which accounts for 10-20% of the total weight of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator.

5. The preparation method according to claim 2, characterized in that: The initiator is azobisisoheptanenitrile, which accounts for 0.1%-0.2% of the total mass of the BMA / IBOMA resin monomer, the crosslinking agent and the initiator.

6. Use of the plastic embedding agent according to claim 1 or the plastic embedding agent obtained by the preparation method according to any one of claims 2 to 5 in shaping and embedding biological tissue samples, wherein the biological tissue samples have fluorescent labels or can emit fluorescence.

7. A plastic embedding method compatible with multicolor fluorescence and immunofluorescence labeling, characterized in that: The plastic embedding method comprises the step of embedding a biological tissue sample having multicolor fluorescence and / or immunofluorescence labeling with the plastic embedding agent described in claim 1 or the plastic embedding agent obtained by the preparation method of any one of claims 2-5.

8. The plastic embedding method according to claim 7, characterized in that: The plastic embedding method comprises the following steps: (1) Pretreatment: Fix the biological tissue sample with a fixative at 4°C for 24-48 hours, then rinse the fixed biological tissue sample with a buffer solution for multiple times, replacing the buffer solution every 3-4 hours; (2) Gradient dehydration: The pretreated biological tissue sample is placed in 50% ethanol precooled at 4°C overnight, and then placed in 75%, 95% ethanol and anhydrous ethanol precooled 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; (3) Gradient infiltration: dilute the plastic embedding agent BMA / IBOMA resin to 50% and 75% concentrations with anhydrous ethanol; place the completely dehydrated biological tissue sample in 50% BMA / IBOMA resin precooled at 4°C overnight, and then place it in 75% and 100% BMA / IBOMA resin for gradient infiltration, each gradient for 1-2 hours; finally, place the biological tissue sample in 100% BMA / IBOMA resin overnight, and then place the biological tissue sample in 100% BMA / IBOMA resin for one day to allow the BMA / IBOMA resin to fully penetrate the biological tissue sample; (4) Polymerization embedding: The infiltrated biological tissue sample is placed in a gelatin capsule, and then 100% BMA / IBOMA resin is slowly added to the capsule to fill the capsule. Finally, thermal polymerization is performed to complete the embedding of the biological tissue sample.

9. The plastic embedding method according to claim 8, characterized in that: In step (1), the fixing solution is 4% paraformaldehyde; the buffer is 0.01M phosphate buffer; and the number of times the biological tissue sample is rinsed with the buffer is three times.

10. The plastic embedding method according to claim 8, characterized in that: The thermal polymerization is carried out in an oven, the polymerization temperature is 38-45° C., and the polymerization time is 6-24 hours.

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