JP6721899B2 - Embedding method for electron microscope mainly composed of highly hydrophilic polymer monomer - Google Patents

Description

TECHNICAL FIELD The present invention relates to an embedding method for an electron microscope, which contains a highly hydrophilic polymer monomer as a formalin substitute solution as a main component.

In the observation area by transmission electron microscopy in immunohistocytochemistry, even if using an existing electron microscope embedding medium such as Lowicryl K4M or LR-White, it may not be observed depending on the biological sample, for example, the type of antibody, Or often very poor results can be obtained.

For example, as an electron staining method for a transmission electron microscope sample, a method is known in which cell tissue embedded in epoxy resin, polymerized, and sliced is immersed in an aqueous solution of uranium acetate, washed, then immersed in a lead staining solution, and then washed. (Patent Document 1). In addition, when embedding a tissue containing water and a radical source in cells, the tissue is immersed in a highly hydrophilic polymer monomer solution containing a crosslinking agent and a polymerization initiation control agent in the highly hydrophilic polymer, A tissue embedding method is also known in which, after nitrogen gas bubbling, the tissue is allowed to stand and polymerize (Patent Documents 2 and 3). However, with these embedding methods, for example, it is difficult to prepare a sample for an ultrathin transmission electron microscope of 100 nm or less, and it is not applicable to staining and observing various antibodies. Therefore, it was necessary to develop a new embedding medium based on a highly hydrophilic resin.

Japanese Patent No. 4292296 Japanese Patent No. 4374435 Japanese Patent No. 4956839

The present invention provides a novel embedding medium based on a highly hydrophilic resin as a formalin substitute solution, and an embedding method for an electron microscope using the embedding medium.

That is, the present invention is as follows.
[1] N-vinyl-2-pyrrolidone, which has high hydrophilicity and becomes a polymer when polymerized, is added to an aqueous solution in which the concentration of the N-vinyl-2-pyrrolidone is 98 to 100%. 1 to 3 parts by weight of a crosslinking agent, 0.0001 to 0.001 parts by weight of a polymerization accelerator, and other monomers as methyl methacrylate, hydroxyethyl methacrylate, dodecyl methacrylate, 2-methyl- 0 to 100 parts by weight selected from the group consisting of 2-propenoic acid, C _12-16 alkyl ester, and (1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl)bismethacrylate. A method for embedding a tissue, which comprises immersing the tissue in an embedding liquid to which the above-mentioned monomer is added, and allowing the tissue to stand to polymerize.
[2] The tissue embedding method according to the above [1], wherein N,N′-methylene-diacrylamide is selected as the crosslinking agent.
[3] The tissue embedding method according to the above [1] or [2], wherein the polymerization accelerator is selected from the group consisting of Qcu-1 and Qcu-3.

The present invention provides an embedding agent containing N-vinyl-2-pyrrolidone (NPV), which is a highly hydrophilic monomer, as a main agent, and a cross-linking agent, a polymerization accelerator, and other monomers such as methacrylmethyl added thereto. By using the embedding medium, a thin sample of 100 nm or less can be obtained inexpensively and easily, and further staining observation using various kinds of antibodies has become possible.

The results of observation under an optical microscope after sensitization with silver (PD method) by reacting with a gold-labeled anti-IgG antibody are shown.

The present invention relates to an embedding method for an electron microscope, which comprises a highly hydrophilic polymer monomer serving as a formalin substitute solution as a main component, and hitherto, sufficient effects were not obtained depending on, for example, the type of antibody to be observed. The technical drawback is solved. Therefore, according to the present invention, the conditions required for the embedding medium used in the electron microscope are that the slice has suitable hardness and elasticity when it is prepared, that the sliced slice floats in water, and that it can withstand an electron beam. Provided are a novel embedding agent based on a highly hydrophilic resin, which satisfies the requirements for various staining, and a method for embedding a biological sample (organ, tissue, etc.) using the embedding agent. ..

More specifically, the present invention uses N-vinyl-2-pyrrolidone, which has high hydrophilicity and becomes a polymer when polymerized, and increases the concentration of the N-vinyl-2-pyrrolidone to 98 to 100%. 1 to 5 parts by weight of a cross-linking agent, 0.0001 to 0.001 parts by weight of a polymerization accelerator, and other monomers such as methyl methacrylate, hydroxyethyl methacrylate, and methacrylic acid in 100 parts by weight of the aqueous solution. At least one selected from the group consisting of acid dodecyl, 2-methyl-2-propenoic acid, C _12-16 alkyl ester, (1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl)bismethacrylate There is provided a method for embedding a tissue, which comprises immersing the tissue in an embedding liquid to which 0 to 100 parts by weight of a monomer is added, and allowing the tissue to stand and polymerize.

(1) Electron Microscope Sample In the present invention, biological samples suitable for the embedding method of the present invention include, but are not limited to, various animal organs, organs, tissues, and cells. Further, in the present invention, pretreatment of the biological sample with formalin (formaldehyde aqueous solution of about 4%) for protein immobilization is not essential. That is, by directly embedding a biological sample in the embedding medium of the present invention, it is possible to prevent a decrease in sensitivity such as inhibition of antibody binding due to the structural change due to protein immobilization. However, if formalin or paraformaldehyde fixation is required in advance for long-term storage of the biological sample or as a premise of using other embedding materials, the fixation treatment may be performed, and even in this case It is possible to apply the embedding method of the present invention.

(2) Embedding agent The embedding agent used in the embedding method of the present invention is N-vinyl-2-pyrrolidone (NVP), which is a highly hydrophilic polymer monomer, and is polymerized under predetermined polymerization conditions. By doing so, the main component is a highly hydrophilic polymer. According to the present invention, a biological sample such as a tissue is embedded, and as an embedding liquid before polymerization, an aqueous solution having a concentration of 98 to 100% and containing 100 parts by weight of the above-mentioned main component is used. 1 to 5 parts by weight of a cross-linking agent, and 0 to 100 parts by weight of a monomer different from the main agent are added to the liquid.

The method for embedding biological material in resin is to dehydrate the material fixed with formalin or the like or the unfixed raw sample with ethyl alcohol, dip it in the embedding resin, infiltrate the resin, and then polymerize it. There is a method in which a sample is diluted with water and immersed in an embedding medium having a low concentration to a high concentration to allow a resin to permeate and then polymerize. In the present invention, the method of dipping and embedding an embedding liquid after dehydration with ethyl alcohol in the preceding paragraph, or the method of dipping the low concentration NVP monomer (50%) into a high concentration NPV monomer (100%) in the following paragraph There is a method of polymerizing by immersing in an embedding liquid. Especially in the following section, when the sample is immersed in NPV monomer at a concentration of less than 50%, water content becomes excessive, resulting in cell destruction. High concentration, especially 100% NVP monomer causes dehydration and contraction of the sample, and the NVP monomer in the sample surface layer portion in a short time. Is polymerized and solidified by the action of the radicals of the material itself, making it difficult to prepare an appropriate electron microscope sample. Therefore, in the present invention, the method of the preceding section was mainly used.

In the present invention, examples of the cross-linking agent added to the embedding liquid are not limited as long as they allow polymerization of the main agent (NVP monomer), and for example, N,N′-methylene-diacrylamide is used. Can be mentioned. The amount of the cross-linking agent added to the NVP aqueous solution is preferably within a range in which the NVP monomer is appropriately cross-linked and polymerized, for example, 1 to 5 parts by weight, more preferably 2 to 3 parts by weight, relative to 100 parts by weight of the NVP aqueous solution. Good. A person skilled in the art can appropriately change the addition amount of the crosslinking agent depending on the type of the crosslinking agent used.

In the present invention, examples of the polymerization accelerator added to the embedding liquid are not limited as long as they allow the main agent (NVP monomer) to be polymerized, and examples thereof include Qcu-1 and Qcu-3. Can be mentioned. The addition amount of the polymerization accelerator in the NVP aqueous solution is preferably within a range in which the polymerization of the NVP monomer is appropriately promoted, and for example, 0.0001 to 0.001 part by weight, more preferably 0. It may be 0004 to 0.0005 parts by weight. A person skilled in the art can appropriately change the addition amount of the polymerization accelerator depending on the type of the polymerization accelerator used.

Further, the embedding liquid may be blended with a monomer other than the NVP monomer in order to adjust the hardness of the resin block after polymerization. Such monomers include, but are not limited to, methyl methacrylate, hydroxyethyl methacrylate, dodecyl methacrylate, 2-methyl-2-propenoic acid, C _12-16 alkyl esters, (1-methylethylidene)bis(4,1 -Phenyleneoxy-2,1-ethanediyl)bismethacrylate may be selected from the group consisting of one or more. Preferred are methyl methacrylate and hydroxyethyl methacrylate. The amount of the above-mentioned monomer added to the embedding liquid may be 0 to 100 parts by weight, more preferably 40 to 60 parts by weight. Those skilled in the art can appropriately change the addition amount of the polymerization accelerator depending on the type of the polymerization accelerator used.

(3) Embedding method The embedding method of the present invention is characterized in that a biological sample is immersed in the embedding agent of the present invention, and allowed to stand to polymerize. The time period for immersing the biological sample in the embedding medium and allowing it to stand still is not limited, but may be at least 10 hours for at least 1 mm in thickness of the sample. 12 hours for 0.5 mm. The polymerization of the monomer can be performed according to general conditions well known in the art. For example, a resin block having desired hardness and elasticity can be obtained by polymerizing the embedding medium of the present invention at 40 to 70° C. for 12 to 72 hours under atmospheric pressure. Preferred polymerization conditions are 58 to 62° C. and 18 to 24 hours under atmospheric pressure.

(4) Evaluation of sample after embedding The antibody reaction to the biological sample embedded by the embedding medium of the present invention was evaluated by preparing a section (for example, 1-2 μm thick) for observation by an optical microscope. Immunohistocytochemistry can be performed by reacting a biological sample with a primary antibody specific to the biological sample and then with a labeled secondary antibody against the primary antibody. For example, when the biological sample is pancreas, IgG such as anti-glucagon antibody and anti-insulin antibody is reacted at an appropriate concentration as a primary antibody, and then IgG-Gold is appropriately used as a labeled secondary antibody for the antibody. It is possible to examine the reactivity of the antibody with respect to the embedded sample by reacting at various concentrations, and then detecting the labeled Gold under an optical microscope.

The following examples illustrate various aspects of this disclosure. It will be apparent to those skilled in the art that numerous modifications to both materials and methods may be made without departing from the scope of this disclosure. All reagents and solvents purchased from commercial suppliers are used without further purification or processing.

Example 1: Examination of embedding agents of various compositions Method N-vinyl-2-pyrrolidone (Wako Pure Chemical Industries, Ltd.), which is a highly hydrophilic monomer, is selected as the main component of the embedding medium, and the cross-linking agent listed in Table 1 below for a predetermined amount of this component, A polymerization accelerator and other monomers were mixed in predetermined amounts, and the properties of each embedding agent were observed. Specifically, as samples, pancreas, liver, spleen, kidney, adrenal gland, pituitary gland, brain, testis, and heart of a male (female 8 to 12-week-old rat (SD strain) (Kudo Co., Ltd.) are first tested. Fixed with 4% paraformaldehyde fixative, embedded in each embedding medium, and sliced with a glass knife to make slices (1-2 μm), and the slices were prepared by changing the ratio of the combination of components of the embedding medium. The condition (hardened state, hardness, reaction to water) and the toluidine blue (Merck) staining were used for evaluation.

Regarding immunostaining, embedded pancreatic sections were stained with two types of anti-insulin antibody and anti-glucagon antibody (DAKO) and evaluated. In addition, with respect to sections of organs such as the liver other than the pancreas, immunostaining was performed for antigens characteristically expressed in each organ. The evaluation was performed by observing the reactivity of the antibody with a transmission electron microscope (Hitachi). The immunostaining was evaluated by comparing all the antibodies with LR-White (London Resinn Co. Ltd) sections.

2. Results Table 1 shows the mixing ratio of each embedding medium.

No. Two molds were prepared for each of Nos. 1 to 7, and the deformation of the mold and the floating property in water and the extensibility of each 1 μm section were evaluated. The results are shown in Table 2.

No. 1, No. 2, and No. Sample No. 3 was evaluated to be inappropriate at the trimming stage because the mold was relatively large and hard and brittle. No. 5, No. 6 and No. 6 In No. 7, the sections were not submerged or extended. Therefore, the deformation of the mold was one of the two, and the No. 2 having the floating property and the extensibility. The embedding medium of No. 4 was adopted in the immunostaining experiment of the following organs.

Example 2: Immunostaining No. 1 evaluated as good in Example 1 After embedding the rat pancreas using the embedding medium of Example 4, immunostaining (IgG-Gold PD method) was carried out by a light microscope for examination. Briefly, prepare a 1-2 μm-thick section, react with a primary antibody against swine glucagon (DAKO), and then react with a gold-labeled anti-IgG antibody, followed by sensitization with silver (PD method), and then using an optical microscope. See below (Figure 1). The resulting dilution adaptation of the anti-porcine glucagon antibody is shown in Table 3.

The immunostaining using the embedding medium of the present invention showed as good results as the conventional embedding method, LR-White, at all the dilution ratios examined.

As another immunostaining, the same test was performed on the mouse pancreas using a primary antibody against insulin. As a result, in the conventional method (LR-White), it was possible to observe and react even when the antibody was diluted to 4000 times, but in the present invention, the same degree of reaction was shown even when diluted to 8000 times or more. When an anti-glucagon antibody was used as the primary antibody for the mouse pancreas, it was 4000 times in the conventional method and 12000 times in the present invention.

The present invention addresses the need for clearly observing antibodies with a transmission electron microscope in immunohistochemistry. Further, the cost when the conventional method is used can be reduced to about 1/4.

Claims (3)

Using N-vinyl-2-pyrrolidone, which has high hydrophilicity and becomes a polymer when polymerized, to an aqueous solution in which the concentration of the N-vinyl-2-pyrrolidone is 98 to 100%, and 100 parts by weight of the aqueous solution is added. 1 to 5 parts by weight of a crosslinking agent, 0.0001 to 0.001 parts by weight of a polymerization accelerator, and other monomers such as methyl methacrylate, hydroxyethyl methacrylate, dodecyl methacrylate, and 2-methyl-2-propene. Acid , 1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl)bismethacrylate in an embedding liquid to which 0 to 100 parts by weight of a monomer selected from the group consisting of 1 or more are added. A method for embedding a tissue, which comprises immersing the tissue, allowing it to stand and polymerizing it. The tissue embedding method according to claim 1, wherein N,N'-methylene-diacrylamide is selected as the cross-linking agent. The tissue embedding method according to claim 1 or 2, wherein the polymerization accelerator is selected from the group consisting of Qcu-1 and Qcu-3.