Laminate, production method for same, preparation method for paraffin section sample, and preparation method for paraffin sample piece

Abstract

The present invention addresses the problem of providing a laminate that is easily manipulated and results in little damage to an original paraffin block sample when a paraffin section sample is prepared, a production method for the laminate, a preparation method for a paraffin section sample, and a preparation method for a paraffin sample piece.　Provided is a laminate that includes a substrate, a first paraffin layer that is provided on at least a portion of the surface of the substrate, and a second paraffin layer that is provided on at least a portion of the first paraffin layer, the melting point T1 of a first paraffin that constitutes the first paraffin layer being higher than the melting point T2 of a second paraffin that constitutes the second paraffin layer.

Description

Laminate and method for manufacturing the same, method for preparing paraffin section specimens, and method for preparing paraffin specimen pieces

[0001] The present invention relates to a laminate, a method for manufacturing the same, a method for preparing a paraffin section specimen, and a method for preparing a paraffin specimen.

[0002] Pathological specimens are prepared by thinly slicing formalin-fixed and paraffin-embedded blocks to a thickness of approximately 3 μm. In pathological diagnosis, it is sometimes necessary to analyze multiple specimens simultaneously. Therefore, a technique called tissue microarray (TMA) is used to combine multiple pathological specimens into a single specimen. This technique involves punching out paraffin blocks and combining the tissue cores into a single paraffin block. A tissue microarray is a paraffin block produced by extracting tissue cores from multiple paraffin blocks and re-embedding them as an array into a single block. This technology makes it possible to analyze many samples simultaneously, resulting in significant savings in time and cost.

[0003] Patent Document 1 relates to a manual set for constructing tissue microarrays, which includes a tissue core punch extractor for extracting cylindrical portions of tissue from a donor block. Patent Document 2 discloses a microscope slide having a paraffin protective coating for biological materials and / or inorganic deposits on the microscope slide.

[0004] International Publication No. 2004 / 000992, International Publication No. 2018 / 228508

[0005] Methods for manufacturing tissue microarrays are known, and they can be manufactured, for example, using the manual tissue microarray construction set described in Patent Document 1. However, a problem arises in manufacturing tissue microarrays because the paraffin block is punched out and the tissue core is extracted, which damages the original paraffin block specimen. Furthermore, advanced technology is required for the production of tissue microarrays.

[0006] The present invention aims to provide a laminate and a method for manufacturing the same, a method for preparing a paraffin section specimen, and a method for preparing a paraffin specimen, which can solve the above problems, cause less damage to the original paraffin block specimen when preparing a paraffin section specimen, and are easy to operate.

[0007] The present invention encompasses the following embodiments: [1] A substrate, a first paraffin layer provided on at least a portion of the surface of the substrate, and a second paraffin layer provided on at least a portion of the first paraffin layer, wherein the melting point T of the first paraffin constituting the first paraffin layer １ However, the melting point T of the second paraffin constituting the second paraffin layer ２ A higher, layered structure.

[0008] [2] The laminate according to [1], wherein the first paraffin layer is colored.

[0009] [3] The laminate according to [1] or [2], wherein the substrate is a glass substrate.

[0010] [4] The laminate according to any one of [1] to [3], wherein the substrate is a substrate having a release layer on a part of its surface.

[0011] [5] A method for preparing a paraffin section specimen, comprising the following steps (1-1) to (1-4) in this order: (1-1) A step of thinly slicing a paraffin-embedded specimen to prepare sections; (1-2) A step of attaching the prepared sections onto the second paraffin layer of the laminate described in [1] or [2]; (1-3) A step of cutting off a portion of the section together with a portion of the first and second paraffin layers to which it is attached to isolate the sample core; (1-4) A step of attaching a portion of the section of the isolated sample core to the surface of a second substrate to place the sample core on the second substrate.

[0012] [6] The method according to [5], further comprising the following step (1-4) after the above step (1-4). (1-5)T ２ More than T １ The process involves heating to a temperature below zero to melt the second paraffin, then cooling to fix the sample core to the second substrate.

[0013] [7] The method according to [6], further including the following steps (1-6) and (1-7) after the step (1-5). (1-6) Heating T １ to a temperature above to melt the first paraffin and the second paraffin. (1-7) Removing the melted first paraffin and second paraffin.

[0014] [8] The method according to any one of [5] to [7], wherein in the step (1-4), the second substrate is a glass substrate subjected to a hydrophilic treatment and is wetted with water.

[0015] [9] A method for preparing a tissue microarray, wherein a plurality of sample cores are arranged on the second substrate by the method including the steps (1-1) to (1-4) according to any one of [5] to [8] in this order.

[0016]

[10] A laminate having a substrate, a first paraffin layer provided on at least a part of the surface of the substrate, and a second paraffin layer provided on at least a part of the first paraffin layer, wherein the melting point T of the first paraffin constituting the first paraffin layer １ is higher than the melting point T of the second paraffin constituting the second paraffin layer. A method for manufacturing a laminate, including the following steps (M1) to (M3) in this order. (M1) Immersing the substrate in the first paraffin melted by heating to a temperature above T, and then pulling it up. (M2) After solidifying the first paraffin, immersing the substrate with the first paraffin adhered thereto in the second paraffin melted by heating to a temperature above T, and then pulling it up. (M3) Solidifying the second paraffin. ２ １ ２

[0017]

[11] The manufacturing method according to

[10] , including the following steps (M0-1) and (M0-2) in this order before the step (M1). (M0-1) Providing a release layer on a part of the surface of the substrate. (M0-2) Applying the first paraffin to a part of the substrate without the release layer.

[0018]

[12] A method for preparing a paraffin specimen slide, comprising the following steps (2-1) to (2-3) in this order. (2-1) A step of sectioning a paraffin-embedded specimen to prepare sections. (2-2) A step of attaching the prepared sections onto the paraffin layer of a laminate having a base material and at least one paraffin layer provided on at least a part of the surface of the base material. (2-3) A step of cutting out a part of the section together with a part of the attached paraffin layer to isolate a sample core.

[0019]

[13] The method for preparing a paraffin specimen slide according to

[12] , wherein the laminate having at least one paraffin layer is a laminate in which a water-based medium is interposed between a base material, a paraffin layer attached to a part of the surface of the base material, and a part where the base material and the paraffin layer are not attached.

[0020]

[14] A laminate comprising a base material, a paraffin layer attached to a part of the surface of the base material, and a water-based medium interposed between a part where the base material and the paraffin layer are not attached.

[0021] According to the present invention, there are provided a laminate and a method for manufacturing the same, a method for preparing a specimen of paraffin sections, and a method for preparing a paraffin specimen slide, which cause less damage to the original paraffin block specimen in preparing a paraffin section specimen and are easy to operate. Specifically, according to the present invention, a specimen of a paraffin section of a desired site can be easily prepared only by performing a normal sectioning operation on a paraffin block specimen, and the damage to the paraffin block specimen can be minimized.

[0022] FIG. 1 shows a hematoxylin and eosin (HE) stained image. FIG. 2 shows an antibody stained image. FIGS. 3(a) to (c) are photographs showing the steps of sandwiching a layered paraffin piece placed on a slide glass with a magnet. FIG. 4 is a diagram schematically showing the steps of Production Example 3. FIG. 5 is a diagram schematically showing the steps of Production Example ⑤.

[0023] [Laminate (X)] The laminate of the present invention (hereinafter also referred to as laminate (X)) comprises a base material, a first paraffin layer provided on at least a part of the surface of the base material, and a second paraffin layer provided on at least a part of the first paraffin layer, wherein the melting point T of the first paraffin constituting the first paraffin layer １ However, the melting point T of the second paraffin constituting the second paraffin layer ２ Higher.

[0024] The laminate (X) of the present invention can be used, for example, to prepare a paraffin section specimen by the method for preparing a paraffin section specimen (1) described later. By providing a two-layer structure of a first paraffin layer and a second paraffin layer having different melting points, the first paraffin layer with a higher melting point maintains hardness, facilitating the handling of the laminate and the sample core to be peeled off, while the second paraffin layer with a lower melting point softens even at relatively low temperatures, forming a smooth surface, and enabling the paraffin section to be adhered to the second substrate with high adhesion.

[0025] <Substrate> The substrate is not particularly limited as long as it is a substrate on which a paraffin layer can be laminated. Examples of substrates include glass substrates and resin substrates. Specific examples of resin substrates include polycarbonate (PC), cycloolefin polymer (COP / COC), polyetherimide (PEI), and polyester (PET).

[0026] In one preferred embodiment of the present invention, the substrate is a glass substrate from the viewpoint of easily peeling off the paraffin layer. In particular, a glass substrate is preferred when the substrate does not have a release layer as described later. As the glass substrate, commercially available microscope slides are preferred from the viewpoint of availability. When using microscope slides as the glass substrate, the material is not limited. For example, water glass (soda-lime glass), white glass (borosilicate glass), half-white glass, etc. are examples. The presence or absence of surface treatment is also not limited. For example, it may have a surface treatment coating such as poly-L-lysine (PLL) or aminosilane (APS), or it may not have any surface treatment. From the viewpoint of economy, it is preferable that it does not have any surface treatment. It may also be a microscope slide that has undergone a cleaning and degreasing treatment called "preclean". The presence or absence of treatment of the edges of the glass is also not limited. Specifically, examples include cut edges with no treatment on the edges, chamfered edges and polished edges. From the viewpoint of safety of operation, polished edges are preferred.

[0027] In one preferred embodiment, the substrate has a release layer on a portion of its surface. If the substrate has a release layer on its surface, it is present only on a portion of the surface. For example, the substrate has a release layer in the center of its surface, but not on the periphery. Having a release layer on a portion of the substrate's surface is preferable because it controls excessive adhesion between the paraffin layer and the substrate, making it easier to isolate the sample core in step (1-3) of the method for preparing a paraffin section specimen described later. The release layer is composed of, for example, a fluororesin such as polytetrafluoroethylene (PTFE), a silicone compound, or the like. The release layer can also be formed using a fluororesin adhesive tape.

[0028] When the substrate has a release layer, for example, a glass substrate or a resin substrate can be used as the substrate. Glass substrates are preferred because, for example, microscope slides are readily available. Resin substrates are preferred from the viewpoint of achieving proper adhesion between the paraffin layer and the substrate.

[0029] The thickness of the substrate is not particularly limited. Preferably it is 200 μm or more and 5000 μm or less, more preferably 500 μm or more, even more preferably 700 μm or more, even more preferably 900 μm or more, even more preferably 950 μm or more, and more preferably 2000 μm or less, even more preferably 1500 μm or less, even more preferably 1100 μm or less, and even more preferably 1050 μm or less.

[0030] <Paraffin layer> The laminate of the present invention comprises a first paraffin layer provided on at least a portion of the surface of the substrate and a second paraffin layer provided on at least a portion of the first paraffin layer.

[0031] (First paraffin layer) The first paraffin layer is provided on at least a portion of the surface of the substrate. Preferably, it is provided on at least a portion of one surface of the substrate. In one preferred embodiment, it is provided on the entire surface of one surface of the substrate.

[0032] The first paraffin layer is preferably colored. The coloring is preferably a color with low brightness and saturation, such as black or blue. Coloring can be achieved by the first paraffin layer containing a coloring agent. The coloring agent is not particularly limited as long as it is a coloring agent that can be used in combination with paraffin. For example, from the viewpoint of availability, coloring agents that are commercially available for candles are preferred as coloring agents. The content of the coloring agent can be appropriately set by those skilled in the art. The first paraffin layer is preferred because it improves the visibility of the area containing tissue fragments in the paraffin section that is attached in step (1-2) of the method for preparing a paraffin section specimen described later.

[0033] The thickness of the first paraffin layer is not particularly limited. Preferably it is 200 μm or more and 5000 μm or less, more preferably 500 μm or more, even more preferably 800 μm or more, even more preferably 900 μm or more, and more preferably 2000 μm or less, even more preferably 1200 μm or less, and even more preferably 1100 μm or less.

[0034] (Second paraffin layer) The second paraffin layer is provided on at least a portion of the first paraffin layer. That is, the second paraffin layer is provided on at least a portion of the surface of the first paraffin layer opposite to the substrate. In one preferred embodiment, it is provided on the entire surface of the first paraffin layer. The thickness of the second paraffin layer is not particularly limited. Preferably it is 20 μm or more and 1000 μm or less, more preferably 50 μm or more, even more preferably 100 μm or more, even more preferably 150 μm or more, and more preferably 500 μm or less, even more preferably 300 μm or less, and even more preferably 250 μm or less.

[0035] In one preferred embodiment, the laminate (X) of the present invention is composed of three layers in which a substrate, a first paraffin layer, and a second paraffin layer are laminated in that order.

[0036] (Melting point) The first paraffin layer is composed of the first paraffin, and the second paraffin layer is composed of the second paraffin. The melting point of the first paraffin is T １ Let the melting point of the second paraffin be T. ２ In this case, the melting point T １ melting point T ２ Higher. Two paraffins with different melting points can be selected; the one with the higher melting point is designated as the first paraffin, and the one with the lower melting point is designated as the second paraffin. Melting point T １ and melting point T ２ Difference T １ -T ２ The temperature is usually 5°C or higher, preferably 5°C to 15°C, and more preferably 8°C to 12°C. Paraffins with a melting point of 47°C to 69°C are generally available. In one embodiment, the melting point T １ The melting point T is preferably 55°C to 65°C, more preferably 58°C to 60°C. ２ The temperature is preferably 47°C to 53°C, and more preferably 48°C to 50°C.

[0037] Melting point T １ melting point T ２The first paraffin layer, composed of higher-grade first paraffin, is hard and easy to handle. In particular, in step (1-3) of the method for preparing paraffin section specimens described later (1), it facilitates the isolation of the isolated sample core from the substrate, maintains its hardness, and makes it easy to handle. Melting point T ２ melting point T １ The second paraffin layer, composed of lower-grade paraffin, softens at lower temperatures. In particular, it improves adhesion to the paraffin section when attaching the paraffin section in step (1-2) of the method for preparing paraffin section specimens described later.

[0038] The melting point of paraffin is measured according to the "Method for Testing Melting Point" described in JIS K 2235:2022 "Petroleum Waxes".

[0039] As the first and second paraffins, commercially available paraffins used as embedding materials for pathological tissues can be used. In particular, commercially available hard paraffins can be used as the first paraffins, and commercially available soft paraffins can be used as the second paraffins.

[0040] The laminate (X) of the present invention can be manufactured, for example, according to the "Method for Manufacturing the Laminate (X)" described later. The laminate (X) of the present invention can be used in the method for preparing paraffin section specimens and the method for preparing tissue microarrays described later.

[0041] [Method for manufacturing the laminate (X)] The method for manufacturing the laminate (X) of the present invention includes the following steps (M1) to (M3) in this order. (M1) The substrate is T １ (M2) The process of immersing in the first paraffin that has been heated and melted as described above, and then removing it. After the first paraffin has solidified, the substrate to which the first paraffin has been attached is T ２ The process of immersing the product in the second paraffin that has been heated and melted, and then removing it (M3) The process of solidifying the second paraffin

[0042] <Step (M0)> As the base material, a base material capable of laminating the paraffin layer described in the "Laminate (X)" column above is used. When a base material having a release layer on a part of its surface is used as the base material, the manufacturing method of the present invention may include a step (M0) of providing the base material before step (M1). Specifically, step (M0) may include a step (M0-1) of providing a release layer on a part of the surface of the base material. The release layer can be provided by known methods such as spray coating or dipping coating using a release agent of a desired form mainly composed of a fluororesin such as polytetrafluoroethylene (PTFE) or a silicone compound. The release layer can also be provided by attaching a fluororesin adhesive tape.

[0043] Step (M0) may further include step (M0-2) of applying the first paraffin to the portion of the substrate that does not have a release layer. The first paraffin can be easily applied by rubbing it on, for example, by molding it into a solid such as a rod. The applied first paraffin becomes one with the paraffin that is applied by immersion in step (M1) described later, forming a first paraffin layer.

[0044] The presence of a release layer is preferable because it controls excessive adhesion between the paraffin layer and the substrate, making it easier to isolate the sample core in step (1-3) of the method for preparing a paraffin section specimen described later. On the other hand, by applying the first paraffin to the portion of the substrate that does not have a release layer, the first paraffin layer can be efficiently attached to the substrate. In this way, by using both the formation of a release layer and the application of the first paraffin to the portion of the substrate that does not have a release layer, it is possible to efficiently control the adhesion and detachment of the first paraffin layer to the substrate.

[0045] <Process (M1)> In process (M1), the substrate is T １ The substrate is immersed in the first paraffin that has been heated and melted as described above, and then removed. It is preferable that the substrate to be used in step (M1) is cooled beforehand. The cooling temperature can be, for example, 0 to 10°C. Because the substrate is cooled beforehand, the first paraffin adheres sufficiently to the substrate, especially if the substrate has a release layer on part of its surface.

[0046] The heating temperature of the first paraffin is the melting point T of the first paraffin. １ The above is true, and from the viewpoint of giving paraffin good fluidity, T is preferred. １ More preferably T １ The temperature is above +7°C. There is no particular upper limit as long as the paraffin does not denature, but the inventors have found that if the heating temperature of the paraffin in this process is too high, when the laminate of the present invention manufactured by the present invention is used to prepare the paraffin section specimen according to the present invention described later, the sample core may adhere to the substrate and become difficult to peel off when cutting the section in step (1-3). Therefore, in order to ensure that the sample core can be easily peeled off from the substrate, the heating temperature of the paraffin is preferably T １ +17°C or lower, more preferably T １ The temperature should be +12°C or lower. Therefore, the heating temperature of the first paraffin is preferably T １ +5℃ or higher and T １ The temperature is +17°C or lower, and more preferably T １ +7℃ or higher and T １ The temperature is +12°C or lower. In one embodiment, when paraffin with a melting point of 58 to 60°C is used as the first paraffin, the heating temperature is usually 60°C to 75°C, preferably 65°C to 70°C. From the viewpoint of ease, heating of the paraffin can be carried out using a commercially available wax warmer. It is preferable that the melted first paraffin contains the coloring agent. The amount of coloring agent can be appropriately determined by those skilled in the art.

[0047] The immersion time of the substrate in the melted first paraffin is preferably 0.5 seconds or more and 1 second or less, more preferably less than 1 second, and even more preferably 0.8 seconds or less, from the viewpoint of obtaining a laminate (X) in which the paraffin layer can be easily peeled off from the substrate.

[0048] After removing the substrate to which the melted first paraffin has adhered, it is preferable to remove the first paraffin from one surface of the substrate before the paraffin solidifies. The paraffin can be removed, for example, by wiping. In another embodiment, by providing a masking layer on the surface of the substrate to which the first paraffin layer is not formed, the paraffin can be easily removed without adhering to the substrate.

[0049] <Process (M2)> In process (M2), after the first paraffin is solidified, the substrate on which the first paraffin layer has been formed is T ２ The first paraffin is then immersed in the second paraffin, which has been heated and melted, and then removed. The solidification of the first paraffin is T １ This can be carried out by standing at a temperature below a certain level (e.g., room temperature). The first paraffin solidifies, and a substrate is obtained in which the first paraffin layer is formed on at least a portion of its surface.

[0050] Next, the substrate on which the first paraffin layer is formed on at least a portion of its surface is subjected to T ２ The second paraffin is heated and melted as described above, and then immersed in it. The heating temperature of the second paraffin is the melting point T of the second paraffin. ２ The above is true, and from the viewpoint of giving paraffin good fluidity, T is preferred. ２ More preferably T ２ The temperature must be above +7°C, and there is no particular upper limit as long as the paraffin does not denature. Here, the heating temperature of the second paraffin is the melting point T of the first paraffin. １ It is preferable that the above conditions are met. This is because, when forming the second paraffin layer on the first paraffin layer, a portion of the paraffin on the surface of the first paraffin layer melts and fuses with the second paraffin layer, resulting in a close bond between the first and second paraffin layers. From this viewpoint, the heating temperature of the second paraffin is preferably T １ +10℃ or higher and T １ The temperature is +30°C or lower, and more preferably T １ +15℃ or higher and T １The temperature is +25°C or lower. In one embodiment, when paraffin with a melting point of 58 to 60°C is used as the first paraffin, the heating temperature of the second paraffin is, for example, 70°C to 90°C, preferably 75°C to 85°C, and more preferably 80°C to 85°C. From the viewpoint of ease, heating of the paraffin can be done using a commercially available wax warmer. From the viewpoint of obtaining a laminate (X) in which the paraffin layer can be easily peeled off from the substrate, the immersion time in the second paraffin is preferably 0.2 seconds to 1 second, and more preferably less than 1 second. After removing the substrate to which the melted second paraffin has adhered, it is preferable to remove the second paraffin from the surface of the substrate opposite to the surface on which the second paraffin layer was formed, before the paraffin solidifies. The removal of the paraffin can be done, for example, by wiping. In another embodiment, by providing a masking layer on the surface of the substrate to which the paraffin layer is not formed, the paraffin can be easily removed without adhering to the substrate.

[0051] <Process (M3)> In process (M3), the second paraffin is solidified. The solidification of the second paraffin is T ２ This can be carried out by standing at a temperature below a certain level (e.g., room temperature). The second paraffin solidifies, and a laminate (X) is obtained having a first paraffin layer provided on at least a portion of the surface of the substrate and a second paraffin layer provided on at least a portion of the first paraffin layer.

[0052] [Method for preparing a paraffin section specimen (1)] The method for preparing a paraffin section specimen of the present invention includes the following steps (1-1) to (1-4) in this order: (1-1) A step of thinly slicing the paraffin-embedded specimen to prepare sections. (1-2) A step of attaching the prepared sections onto the second paraffin layer of the laminate (X). (1-3) A step of cutting out a portion of the section together with a portion of the first paraffin layer and the second paraffin layer to which it is attached to isolate the sample core. (1-4) A step of attaching a portion of the section of the isolated sample core to the surface of the second substrate and placing the sample core on the second substrate.

[0053] Except for the steps where a specific temperature is designated, it is preferable to carry out the following steps at room temperature (20°C to 30°C, preferably 25°C to 28°C).

[0054] <Step (1-1)> In Step (1-1), the paraffin-embedded specimen is sliced ​​to prepare sections. (Paraffin-embedded specimen) The paraffin-embedded specimen is prepared by a method known to those skilled in the art. It is usually obtained by fixing tissue taken from a patient with formalin, dehydrating, dealcoholizing, and paraffin infiltration. (Slicing) Slicing of the paraffin-embedded specimen is done by cutting the paraffin-embedded specimen using a device such as a microtome. The thickness of the resulting section is usually between 2 μm and 4 μm, preferably around 3 μm.

[0055] <Step (1-2)> In step (1-2), the section prepared in step (1-1) is attached to the second paraffin layer of the laminate (X) of the present invention. Step (1-2) can be carried out in accordance with the method of attaching paraffin sections to a glass slide in the preparation of a normal tissue specimen, except that the laminate (X) of the present invention is used. An example is described in detail below. First, the section is floated in a liquid such as water to spread it out. Here, the temperature of the liquid such as water is preferably around room temperature, more preferably 20°C to 30°C, and even more preferably 25°C to 28°C. Next, the section that has been spread out by floating in the liquid such as water is placed on the second paraffin layer of the laminate (X) of the present invention in the liquid. Next, it is removed from the liquid and heated to further spread the section. The heating temperature is preferably 40°C to 50°C, more preferably 43°C to 47°C. The heating time is preferably 15 minutes to 90 minutes, more preferably 30 minutes to 60 minutes. This heating and spreading operation can be carried out using a known hot plate for spreading paraffin sections. Next, the sections are heated and dried. The heating temperature is preferably 35°C to 40°C, more preferably 36°C to 38°C. The heating time is preferably overnight, more preferably 12 hours to 20 hours, and even more preferably 15 hours to 18 hours. This heating and spreading operation can be carried out using a known hot plate for spreading paraffin sections, a Taher-type paraffin melter, etc.

[0056] When the first paraffin layer is colored, there is a contrast difference between it and the section, which is almost colorless and transparent, and this is preferable from the viewpoint of section visibility.

[0057] Thus, the section adheres to the second paraffin layer of the laminate (X) of the present invention, and a sample is obtained in which the substrate, the first paraffin layer, the second paraffin layer, and the section are laminated in that order.

[0058] It is preferable to prepare a reference section sample using the section adjacent to the above section by the same procedure as in step (1-2).

[0059] <Step (1-3)> In step (1-3), a portion of the section is cut off together with a portion of the first and second paraffin layers to which it is attached, and the sample core is isolated. (Sample Core) The shape and size of the sample core can be determined by a person skilled in the art according to the size and purpose of the paraffin-embedded specimen. The shape of the sample core is preferably approximately cylindrical. If the shape is cylindrical, the diameter is preferably 1 mm or more and 20 mm or less. The sample core is a laminated piece mainly composed of paraffin, with the first paraffin layer, the second paraffin layer, and the section stacked in that order.

[0060] (Cutting) The sample core is obtained by cutting out a desired portion of the section, along with a portion of the first and second paraffin layers attached to it, from the sample obtained in step (1-2). The cutting position can be appropriately selected by a person skilled in the art. Preferably, the structure of the reference section sample can be visualized by hematoxylin-eosin staining (HE staining), and the cutting position of the sample core can be selected based on this. The sample core can be cut out using a cylindrical instrument, such as a punch used for processing leather products.

[0061] By using the laminate (X) of the present invention described above, the second paraffin layer can be used to attach sections, and the first paraffin layer can be easily peeled off from the substrate. Therefore, a portion of the section can be cut out together with the attached first and second paraffin layers, and the sample core can be isolated.

[0062] <Step (1-4)> In step (1-4), a portion of the section of the isolated sample core is attached to the surface of the second substrate, and the sample core is placed on the second substrate. (Second Substrate) As the second substrate, a substrate such as a glass substrate or a resin substrate can be used. As the second glass substrate, commercially available slide glass that can be used to prepare ordinary paraffin section sample slide plates is preferred from the viewpoint of availability. When using slide glass as the second substrate, the material is not limited. For example, water glass (soda-lime glass), white glass (borosilicate glass), half-white glass, etc. are examples. The slide glass preferably has a hydrophilic surface treatment coating such as poly-L-lysine (PLL) or aminosilane (APS), and more preferably is an aminosilane-coated slide glass. Whether or not the edges of the glass are treated is also not limited. Specifically, examples include cut edges with no treatment on the edges, chamfered edges, and polished edges. From the viewpoint of safety of operation, polished edges are preferred.

[0063] (Placement) A paraffin section specimen is obtained by directly attaching a portion of the section of the isolated sample core to the surface of the second substrate and placing it on the second substrate. The number of sample cores to be placed may be one or more than one. A tissue microarray is prepared by placing multiple sample cores on the second substrate.

[0064] When using a hydrophilic glass substrate as the second substrate, it is preferable that the hydrophilic glass substrate is pre-moistened with water. Moistening the hydrophilic glass substrate with water improves the adhesion between the section and the second substrate.

[0065] <Steps (1-5)> The method for preparing paraffin section specimens of the present invention preferably further includes the following steps (1-5) after steps (1-4). (1-5)T ２ More than T １ The heating temperature in step (1-5) is T, which is used to heat the second paraffin to a temperature below T to melt it, then cool it to fix the sample core to the second substrate. ２ More than T １The temperature is less than 50°C, preferably between 45°C and 50°C. The second paraffin does not need to melt completely in this step; it only needs to soften to the extent that its adhesiveness improves. The heating time in steps (1-5) is preferably between 30 minutes and 2 hours, more preferably between 45 minutes and 1.5 hours. Cooling can be carried out by leaving it at room temperature. ２ More than T １ By heating the second paraffin to a temperature below 2°C to melt it, and then cooling it, the sample core can be fixed to the second substrate.

[0066] Steps (1-5) are preferably performed with the sample core pressed and compressed against the second substrate, from the viewpoint of improving the adhesion of the sample core to the second substrate. The means of pressing are not particularly limited. For example, it can be done by placing magnets on the surface on which the sample core is placed and on the back surface of the second substrate so that they attract each other, and then sandwiching the sample core between them. When using magnetic force, for example, a neodymium magnet with strong magnetic force can be used. Furthermore, its shape and size are not limited. For example, a person skilled in the art can appropriately set them according to the shape and size of the sample core. For example, if the shape of the sample core is substantially cylindrical, a substantially circular magnet can be used, and its diameter is preferably about the same as or larger than the diameter of the sample core, and more preferably 1 mm to 5 mm larger than the diameter of the sample core. By performing steps (1-5) while sandwiching the sample core with magnets, the entire surface of the sample core can be compressed evenly.

[0067] <Step (1-6)> <Step (1-7)> The present invention's method for preparing paraffin section specimens preferably further includes the following steps (1-6) and (1-7) in this order after step (1-5). (1-6)T １ (1-7) A step of heating to melt the first paraffin and the second paraffin (1-7) A step of removing the melted first paraffin and the second paraffin

[0068] The heating temperature in process (1-6) is T １The above is true, preferably 60°C to 70°C, more preferably 62°C to 68°C. Steps (1-6) and (1-7) remove excess paraffin and improve the adhesion between the section and the second substrate.

[0069] Thus, a paraffin-embedded section specimen is prepared. When multiple sample cores are arranged, a tissue microarray is prepared. Paraffin-embedded specimens are rare, and punching out sample cores from the paraffin-embedded specimen itself causes significant damage, which is undesirable from the standpoint of effective utilization of the specimen. According to the method for preparing a paraffin-embedded section specimen (1) of the present invention, damage to the paraffin-embedded specimen is kept to a minimum as it is limited to the normal thin-sectioning process, and a paraffin-embedded section specimen of the desired area can be easily prepared. The obtained specimen or tissue microarray can be subjected to deparaffinization and immersion treatment in accordance with conventional methods, followed by various staining treatments.

[0070] [Method for preparing paraffin specimen pieces] The present invention also provides a method for preparing paraffin specimen pieces comprising the following steps (2-1) to (2-3) in this order: (2-1) A step of thinly slicing a paraffin-embedded specimen to prepare sections. (2-2) A step of attaching the prepared sections to a substrate and a paraffin layer of a laminate (Y) having at least one paraffin layer provided on at least a part of the surface of the substrate. (2-3) A step of cutting off a portion of the section together with a portion of the attached paraffin layer to isolate the sample core.

[0071] Step (2-1) can be carried out in the same manner as step (1-1) of the "Method for preparing paraffin section specimens" described above.

[0072] Step (2-2) can be carried out in the same manner as step (1-2) of the "Method for Preparing Paraffin Section Specimens," except that a laminate (Y) having at least one paraffin layer is used instead of the laminate (X). The laminate (Y) can be manufactured by omitting the formation of the second paraffin layer in the manufacturing method of the laminate (X). Alternatively, the laminate (Y) may be the laminate (X).

[0073] Step (2-3) can be carried out in the same manner as step (1-3) of the "Method for preparing paraffin section specimens" described above.

[0074] The paraffin specimen prepared by the paraffin specimen preparation method of the present invention can be used for nucleic acid analysis, for example, by a method further comprising the following steps (2-4): (2-4) Extracting nucleic acids from the isolated sample core and subjecting it to nucleic acid analysis.

[0075] In step (2-4), nucleic acids are extracted from the sample core isolated in step (2-3) and subjected to nucleic acid analysis. Nucleic acid extraction from the sample core can be performed by known methods such as phenol-chloroform extraction. The extracted nucleic acid may be either DNA or RNA. Examples of nucleic acid analysis using the extracted nucleic acid include PCR, RT-PCR, quantitative RT-PCR, and nucleic acid sequencing.

[0076] According to the present invention's method for preparing paraffin specimens, paraffin specimens for nucleic acid analysis can be obtained with minimal damage to the original paraffin block specimen.

[0077] As the laminate (Y), a laminate (Y-1) manufactured by a method including the following steps (MY-1) to (MY-4) can be used. (MY-1) A step of applying paraffin to a part of the surface of a substrate. (MY-2) A step of forming a water film on the surface of the substrate on which paraffin has been applied to a part of the surface using an aqueous medium. (MY-3) A step of immersing the substrate on which the water film has been formed in heated and melted paraffin, and then pulling it out. (MY-4) A step of solidifying the paraffin.

[0078] Step (MY-1) can be carried out in the same manner as step (M0-2) of the "Method for Manufacturing Laminate (X)" described above. As the substrate, a substrate on which the paraffin layer described in the "Laminate (X)" section above can be laminated is used. Examples of substrates include glass substrates and resin substrates, and a glass substrate is preferred from the viewpoint of being able to easily peel off the paraffin layer. By applying paraffin to a part of the surface of the substrate, sufficient adhesion between the substrate and paraffin can be achieved even if a water film, described later, is interposed.

[0079] In step (MY-2), a water film is formed on the surface of the substrate, which was partially coated with paraffin in step (MY-1), using an aqueous medium. As the aqueous medium, for example, an aqueous solution of dimethyl sulfoxide (DMSO) of about 1-10% (v / v), preferably about 5% (v / v), can be used. The water film can be formed, for example, by immersing the substrate in the aqueous medium and then removing it. By forming a water film, excessive adhesion between the paraffin layer and the substrate is controlled, making it possible to more easily isolate the sample core in step (2-3). In particular, it is thought that using an aqueous solution of DMSO can lower the surface tension, form a more uniform water film, and suppress the evaporation of water.

[0080] In step (MY-3), the substrate on which a water film has been formed in step (MY-2) is immersed in heated and melted paraffin, and then removed. It is preferable to perform this step without delay from step (MY-2) so that a sufficient water film remains. Any type of paraffin can be used. The heating temperature of the paraffin should be above the melting point of the paraffin. For ease of use, the heating of the paraffin can be done using a commercially available wax warmer. It is preferable that the melted paraffin contains the coloring agent. The type and content of the coloring agent can be appropriately determined by those skilled in the art. The color of the coloring agent is preferably a color with low brightness and saturation, such as black or blue. The immersion time in the paraffin is preferably 0.2 seconds or more and 1 second or less, more preferably less than 1 second, and even more preferably 0.5 seconds or less, from the viewpoint of obtaining a laminate (Y-1) in which the paraffin layer can be easily peeled off from the substrate. After removing the substrate to which the molten paraffin has adhered, it is preferable to remove the paraffin from the surface of the substrate opposite to the surface where the paraffin layer was formed, before the paraffin solidifies. The paraffin can be removed, for example, by wiping. In another embodiment, by providing a masking layer on the surface of the substrate where the paraffin layer is not to be formed, the paraffin can be prevented from adhering to the substrate and easily removed.

[0081] In step (MY-4), the paraffin is solidified. The paraffin can be solidified by leaving it to stand at a temperature below its melting point (e.g., room temperature).

[0082] A laminate (Y-1) is produced by a method including the above steps (MY-1) to (MY-4), in which a substrate, a paraffin layer adhering to a part of the surface of the substrate, and an aqueous medium interposed in the portion where the substrate and the paraffin layer do not adhere.

[0083] The present invention will be specifically described below with reference to examples and test cases, but the present invention is not limited in any way to these examples and test cases.

[0084] Manufacturing Example 1: Fabrication of Laminate (1) The following two types of paraffin were used: Hard paraffin: "Paravet 60 GR" (Muto Chemical Co., Ltd., product code 43257; melting point 58-60°C) Soft paraffin: "Paraffin (mp. 48-50°C)" (Fujifilm Wako Pure Chemical Industries, Ltd., product code 162-13385; melting point 48-50°C)

[0085] Two "pro-wax 100" wax warmers (manufactured by Moise Co., Ltd.) were used to heat and melt the two types of paraffin mentioned above. Specifically, paraffin (approximately 300g) was added to about 80-90% of the melting pot's capacity, and after heating and melting the paraffin, the hard paraffin was maintained at around 65-70°C, and the soft paraffin at around 80°C.

[0086] Approximately 25 mL of the black coloring agent "Candle Liquid Color Black" (manufactured by mono Co., Ltd.) was added to the dissolved hard paraffin to color it.

[0087] A microscope slide "Preclean Water-Edged Polished t1.0" (manufactured by Matsunami Glass Industry Co., Ltd., product code S072130, part number S7213, size 76 x 26 mm, thickness 0.9-1.2 mm) was immersed in colored hard paraffin maintained at approximately 70°C and immediately removed. The immersion time was less than 1 second. The paraffin on the back was wiped off. The slide was left to stand at room temperature to allow the colored hard paraffin to solidify.

[0088] After the colored hard paraffin had solidified, it was immersed in soft paraffin maintained at approximately 80°C and immediately removed. The immersion time was less than 1 second. The paraffin on the back was wiped off. It was left to stand at room temperature to allow the soft paraffin to solidify.

[0089] The thickness of the colored hard paraffin layer on the glass slide was approximately 1 mm, and the thickness of the soft paraffin layer was approximately 250 μm.

[0090] Manufacturing Example 2 Preparation of Paraffin Section Specimens Paraffin block samples for pathological diagnosis derived from invasive ductal carcinoma (IDC) in breast cancer patients were sectioned to 3 μm thickness using a microtome (REM-700 lithatome, manufactured by Yamato Koki Kogyo Co., Ltd.). The obtained sections were floated in water at approximately 27°C and spread, and then placed in water onto the soft paraffin layer of the laminate obtained in Manufacturing Example 1. The laminate with the sections was heated and dried using a paraffin spreading machine (Tissue-Tec, manufactured by Sakura Finetech Japan Co., Ltd.) set to 45°C to further spread the sections. The laminate with the sections was placed in a staining basket and dried overnight in a Tacher-type paraffin melter (manufactured by Hirasawa Co., Ltd.) set to 37°C.

[0091] Using a 5mm leatherworking hole punch (made by Bviban), layered paraffin pieces consisting of thin sections, a soft paraffin layer, and a colored hard paraffin layer were cut from the laminate on which the sections were placed.

[0092] The thin section side of the obtained layered paraffin piece was moistened with water, and the thin section side was attached to a microscope slide glass "New Silane 3 Star Frost Water-Edged 20F" (Muto Chemical Co., Ltd., product code 517628). An 8 mm diameter neodymium magnet (ELECFIND Co., Ltd.) was placed on the opposite side of the attached layered paraffin piece. Another 8 mm diameter neodymium magnet was placed so as to sandwich the layered paraffin piece and the microscope slide glass, attracting it to the magnet on the opposite side. Then, with the slide glass and section sandwiched between the neodymium magnets, the slide glass was placed on a hot plate set to 45°C to soften the soft paraffin and make the section adhere tightly. After that, it was left to stand at room temperature for about 1 hour. Figure 3 shows the steps of attaching the thin section side to the microscope slide glass (Figure 3(a)), placing a neodymium magnet on the opposite side (Figure 3(b)), and sandwiching it with another neodymium magnet (Figure 3(c)).

[0093] Afterward, the neodymium magnets were removed, and the specimens were placed in an oven set to 65°C to melt and remove excess hard and soft paraffin. This process caused the sections to adhere to the glass slide in a way that made them appear as if they were baked onto it.

[0094] Example 1 (HE staining) The paraffin section specimens obtained in Production Example 2 were stained with hematoxylin and eosin (HE) using an automated staining device, Tissue-Tek Prisma (manufactured by Sakura FineTech Japan Co., Ltd.). The specific procedure is as follows. (i-1) Deparaffinization process: Xylene (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code: 249-00097) 3 baths (5 minutes each) (i-2) Dexyleneization process: 100% alcohol (Ethanol 100 solvent for pathological staining, Muto Chemical Co., Ltd., product code: 43105) 3 baths (1 minute each) (i-3) Immersion process: 70% alcohol 1 bath (1 minute) (i-4) Running water rinsing process: Running water rinsing 1 minute (i-5) Water rinsing process: Deionized water 1 bath (1 minute) (i-6) Staining process: Meyer hematoxylin solution (1.5x dilution method) 1 bath (5 minutes) (i-7) Running water rinsing process: Running water rinsing 10 minutes (i-8) Water rinsing process: Deionized water 1 bath (30 seconds) (i-9) Dehydration process: 95% alcohol 1 bath (1 minute) (i-10) Staining process: Eosin staining solution, 1 bath (3 minutes) (i-11) Dehydration process: 100% alcohol, 3 baths (1 minute each) (i-12) Clearing process: Xylene, 2 baths (2 minutes each) (i-13) Clearing process: Xylene, 1 bath (1 minute)

[0095] Finally, the specimens were mounted using an automated glass mounting system, Tissue-Tek Glass g2 (manufactured by Sakura Finetech Japan Co., Ltd.). The HE-stained image is shown in Figure 1.

[0096] (Antibody Staining) The paraffin sections obtained in Production Example 2 above were antibody-stained using the automated staining system Dako Omnis (manufactured by Agilent Technologies) and the immunoglobulin kit for tissue examination Dako EnVision FLEX DAB+ and chromogenic substrate / kit (for Dako Omnis) (manufactured by Agilent Technologies). The specific procedure is as follows. Note that the sections stained with HE and the sections stained with antibody were serial sections. (ii-1) Deparaffinization step: Clearify Cleaning Agent (Agilent Technologies) 1 tank (1 minute) (ii-2) Thermal activation step: Target Retrieval Solution, High pH (included in the kit) 1 tank (97°C, 30 minutes) (ii-3) Blocking step: Blocking reagent (included in the kit) 1 tank (3 minutes) (ii-4) Primary antibody reaction step: Monoclonal Mouse Anti-Human Progesterone Receptor Clone PgR 1294 (Agilent Technologies) 10 minutes (ii-5) Polymer reagent reaction step: Polymer reagent (included in the kit) 1 tank (20 minutes) (ii-6) Chromogenic step: 1 tank (5 minutes) of the chromogenic substrate and chromogenic buffer provided in the kit (ii-7) Nuclear staining step: 8 minutes of Dako Omnis hematoxylin (for Dako Omnis) (manufactured by Agilent Technologies) (ii-8) Dehydration step: 3 tanks (1 minute each) of 100% alcohol (ii-9) Clearing step: 2 tanks (2 minutes each) of xylene (ii-10) Clearing step: 1 tank (1 minute) of xylene

[0097] Finally, the samples were mounted using an automated glass mounting system, Tissue-Tek Glass g2 (manufactured by Sakura FineTech Japan Co., Ltd.). The antibody stained image is shown in Figure 2.

[0098] Manufacturing Example 3: Fabrication of Laminate (2) The same two types of paraffin as in Manufacturing Example 1 were used.

[0099] A fluororesin adhesive tape (Chukoh Flow ASF-110FR, manufactured by Chukoh Chemical Industries, Ltd.) was applied to the center of the surface of a frosted microscope slide to coat the central part of the slide. The fluororesin adhesive tape was not applied to the areas 3 mm wide from the long side of the slide and 5 mm wide from the short side opposite the frosted area. Solidified hard paraffin was rubbed and applied to the areas where the fluororesin adhesive tape was not applied. Masking tape (243J Plus, manufactured by 3M) was applied to the entire back surface of the microscope slide.

[0100] Two "pro-wax 100" wax warmers (manufactured by Moise Co., Ltd.) were used to heat and melt the two types of paraffin mentioned above. Specifically, paraffin (approximately 300g) was added to about 80-90% of the melting pot's capacity, and after heating and melting the paraffin, the hard paraffin was maintained at around 65-70°C, and the soft paraffin at around 80°C.

[0101] Approximately 25 mL of the black coloring agent "Candle Liquid Color Black" (manufactured by mono Co., Ltd.) was added to the dissolved hard paraffin to color it.

[0102] A microscope slide was immersed in colored hard paraffin maintained at approximately 70°C and immediately removed. The immersion time was less than 1 second. The slide was then left to stand at room temperature to allow the colored hard paraffin to solidify.

[0103] After the colored hard paraffin had solidified, it was immersed in soft paraffin maintained at approximately 80°C and immediately removed. The immersion time was less than 1 second. The soft paraffin was allowed to solidify at room temperature. The masking tape was removed, and any excess paraffin was removed.

[0104] Figure 4 schematically shows the process of manufacturing example 3.

[0105] Manufacturing Example 4: Preparation of Paraffin Section Specimens (2) Paraffin section specimens were prepared in the same manner as in Manufacturing Example 2, except that the laminate obtained in Manufacturing Example 3 was used. Layered paraffin sections could be easily cut out using a hole punch.

[0106] Manufacturing Example 5: Fabrication of Laminate (3) The following paraffin was used: "Paravet 60 GR" (Muto Chemical Co., Ltd., product number 43257; melting point 58-60°C)

[0107] The paraffin was heated and melted using a wax warmer "pro-wax 100" (manufactured by Moise Co., Ltd.). Specifically, paraffin was added to about 80-90% of the melting pot's capacity (approximately 300g), heated until the paraffin melted, and then maintained at around 95-100°C.

[0108] Approximately 5g of the black, lipid-soluble dye Zudan Black B (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the dissolved hard paraffin to color it.

[0109] 150 mL of pure water was placed in a slide staining bath (manufactured by Matsunami Glass Industry Co., Ltd., product number BT-210, external dimensions 50 x 100 x 100 mm), and 7.5 mL of dimethyl sulfoxide (DMSO) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to achieve a final concentration of 5% (v / v).

[0110] A paraffin tip was rubbed against the frosted side and the opposite edge of a microscope slide "Starfrost Water-Edged (White) 20F Blood" (manufactured by Muto Chemical Co., Ltd., product number 511620, size 76 x 26 x 1.0 mm, frosted 20 mm).

[0111] Next, the microscope slide was immersed in a 5% (v / v) DMSO aqueous solution up to near-frost. Furthermore, immediately after removing the slide from the solution, while the water film was still present, it was immersed in paraffin maintained at approximately 100°C and immediately removed. The immersion time was less than 0.5 seconds.

[0112] The microscope slide was placed face up on a paper towel. After about 20-30 seconds, the paraffin began to solidify. While it was still soft, the slide was lifted and the paraffin adhering to the back was peeled off. It was then left to stand at room temperature to allow the colored paraffin to solidify. The thickness of the colored paraffin layer on the microscope slide was approximately 0.18-0.25 mm.

[0113] Figure 5 schematically shows the process of manufacturing example 5.

[0114] Preparation Example 6: Preparation of Paraffin Section Specimens (3) Sections of the paraffin block sample were floated in water at approximately 27°C and spread out. The sections were then placed in water on the paraffin layer of the laminate obtained in Preparation Example 5. The outer periphery of the target area was scraped with sharp tweezers, and the paraffin of the target region was subjected to nucleic acid extraction.

Claims

1. A material comprising a base material, a first paraffin layer provided on at least a portion of the surface of the base material, and a second paraffin layer provided on at least a portion of the first paraffin layer, wherein the melting point T of the first paraffin constituting the first paraffin layer １ However, the melting point T of the second paraffin constituting the second paraffin layer ２ A higher, layered structure.

2. The laminate according to claim 1, wherein the first paraffin layer is colored.

3. The laminate according to claim 1, wherein the substrate is a glass substrate.

4. The laminate according to claim 1, wherein the substrate is a substrate having a release layer on a part of its surface.

5. A method for preparing a paraffin section specimen, comprising the following steps (1-1) to (1-4) in this order: (1-1) A step of thinly slicing a paraffin-embedded specimen to prepare sections; (1-2) A step of attaching the prepared sections onto the second paraffin layer of the laminate described in claim 1; (1-3) A step of cutting off a portion of the sections together with a portion of the attached first paraffin layer and a portion of the second paraffin layer to isolate the sample core; (1-4) A step of attaching a portion of the section of the isolated sample core to the surface of a second substrate to place the sample core on the second substrate.

6. The method according to claim 5, further comprising the following step (1-4) after the above step (1-4). (1-5)T ２ More than T １ The process involves heating to a temperature below zero to melt the second paraffin, then cooling to fix the sample core to the second substrate.

7. The method according to claim 6, further comprising the following steps (1-6) and (1-7) after the above step (1-5). (1-6)T １ (1-7) A step of heating to melt the first paraffin and the second paraffin (1-7) A step of removing the melted first paraffin and the second paraffin 8. The method according to claim 5, wherein in step (1-4) the second substrate is a hydrophilized glass substrate and is wetted with water.

9. A method for preparing a tissue microarray, comprising arranging a plurality of sample cores on a second substrate by a method comprising the steps (1-1) to (1-4) described in claim 5 in that order.

10. A laminate having a substrate, a first paraffin layer provided on at least a part of the surface of the substrate, and a second paraffin layer provided on at least a part of the first paraffin layer, wherein the melting point T １ of the first paraffin constituting the first paraffin layer is higher than the melting point T ２ of the second paraffin constituting the second paraffin layer, and a method for producing the laminate, the method including the following steps (M1) to (M3) in this order. (M1) A step of immersing the substrate in the first paraffin heated to T １ or higher and melted, and then pulling it up. (M2) After solidifying the first paraffin, a step of immersing the substrate with the first paraffin attached therein in the second paraffin heated to T ２ or higher and melted, and then pulling it up. (M3) A step of solidifying the second paraffin 11. The manufacturing method according to claim 10, comprising the following steps (M0-1) and (M0-2) in this order before step (M1): (M0-1) A step of providing a release layer on a part of the surface of the substrate; (M0-2) A step of applying the first paraffin to the part of the substrate that does not have a release layer.

12. A method for preparing a paraffin specimen, comprising the following steps (2-1) to (2-3) in this order: (2-1) A step of thinly slicing a paraffin-embedded specimen to prepare sections; (2-2) A step of attaching the prepared sections onto a base material and a paraffin layer of a laminate having at least one paraffin layer provided on at least a part of the surface of the base material; (2-3) A step of cutting off a portion of the section together with a portion of the attached paraffin layer to isolate the specimen core.

13. The method for preparing a paraffin specimen according to claim 12, wherein the laminate having at least one paraffin layer comprises a base material, a paraffin layer adhering to a part of the surface of the base material, and an aqueous medium interposed in the portion where the base material and the paraffin layer do not adhere.

14. A laminate comprising a substrate, a paraffin layer adhering to a portion of the surface of the substrate, and an aqueous medium interposed in the portion where the substrate and the paraffin layer do not adhere.

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