Method for producing embedded object, method for producing sample, centrifugal tube for producing embedded object, and kit for producing embedded object

Abstract

The present invention addresses the problem of: providing a method for producing an embedded object, the method enabling an embedded object to be easily obtained without cell loss by embedding a target cell, etc., in a capsule produced from agarose; providing a method for producing a sample in which the embedded object is used; and providing a centrifugal tube for producing an embedded object, the centrifugal tube being used for producing the embedded object, and a kit for producing an embedded object, the kit including the centrifugal tube.　As a means for solving such a problem, there is provided a method for producing an embedded object of a target object, the method having the following steps 1 to 6 in this order.　Step 1: a step for preparing a centrifugal tube having therein a recessed part formed of agarose gel. Step 2: a step for adding, to the centrifugal tube, a suspension containing a target object to be embedded. Step 3: a step for centrifuging the suspension in the centrifugal tube, and causing the target object to be embedded to settle at the bottom of the recessed part formed of agarose gel. Step 4: a step for removing a supernatant. Step 5: a step for adding, to the centrifugal tube, agarose gel melted by heating, and then cooling and re-curing the agarose gel. Step 6: a step for taking out an embedded object of the target object, in which the target object is embedded in the agarose gel, from the centrifugal tube.

Description

Method for producing an embedded specimen, method for producing a specimen, centrifuge tube for producing an embedded specimen, and kit for producing an embedded specimen

[0001] The present invention relates to a method for producing an embedded specimen, a method for producing a specimen, a centrifuge tube for producing an embedded specimen, and a kit for producing an embedded specimen.

[0002] A cell block is a specimen produced using cell specimens aggregated by various methods, and since thin sections can be produced in the same manner as conventional tissue specimens by producing a formalin-fixed paraffin block, it can be used not only for general hematoxylin-eosin staining, special staining, immunohistochemistry (IHC) method, in situ hybridization (ISH) method, but also for gene-related tests (gene analysis tests). Cell blocks can be applied not only to cell specimens obtained from body fluids, puncture fluids, cytological specimens, etc., but also to cell lines, microtissues, etc.

[0003] Patent Document 1 aims to provide a method for producing a cytological specimen that can easily produce a cytological specimen that can be stored for a long time by performing centrifugation. The method includes a step of filtering a cytological specimen containing a test cell through a filter having a pore size smaller than the size of the test cell and attaching the test cell to a predetermined region of the filter, a step of embedding the filter to which the test cell is attached with an embedding agent to produce an embedding agent block, and a step of producing a thin section specimen by thinly slicing the obtained embedding agent block in a direction in which a cross section of the region of the filter to which the test cell is attached appears on the surface of the thin section specimen. Non-Patent Document 1 discloses a transparent hollow agarose gel tube for performing live imaging of embryos.

[0004] Japanese Patent Application Laid-Open No. 2006-208317

[0005] Udan, R.S. et al. Development, vol.141, issue 22, pages 4406-4414, 2014

[0006] The method described in Patent Document 1 has the problem of difficulty in aggregating a sufficient amount of cells, and also of cell dispersal during the embedding process. Conventional methods for producing cell blocks have made it difficult to form blocks of cells collected by centrifugation without causing them to break down. Methods have been proposed to collect cells collected by centrifugation while preventing their breakdown with various gel-like materials, but in conventional methods, materials that interfere with various stains are interposed between cells, so there has been a strong desire for the development of a technology to produce cell blocks consisting of aggregated cells without the interposition of materials.

[0007] The present invention aims to provide a method for preparing an embedding material, which allows for the simple production of an embedding material without cell loss by embedding target cells or the like in a capsule made of agarose. It also aims to provide a method for preparing a specimen using the aforementioned embedding material. Furthermore, the present invention aims to provide a centrifuge tube for preparing the embedding material and an embedding material preparation kit including the centrifuge tube used in the preparation of the aforementioned embedding material.

[0008] The present invention relates to the following <1> to <8>. <1> A method for preparing an embedded object comprising the following steps 1 to 6 in this order: Step 1: Prepare a centrifuge tube having a recess formed of agarose gel inside Step 2: Add a suspension containing the object to be embedded to the centrifuge tube Step 3: Centrifuge the suspension in the centrifuge tube to allow the object to be embedded to settle at the bottom of the recess formed of agarose gel Step 4: Remove the supernatant Step 5: Add agarose gel melted by heating to the centrifuge tube, then cool and reharden the agarose gel, and Step 6: Remove the embedded object from the centrifuge tube in which the object has been embedded in the agarose gel <2> The method for preparing an embedded object according to <1>, wherein the object to be embedded is a fixed cell or tissue fragment. <3> The method for preparing an embedded object according to <1> or <2>, wherein the centrifugation in step 3 is performed by a swing rotor type centrifuge. <4> A method for preparing a specimen, comprising the steps of: preparing a paraffin-embedded block by embedding an embedding material obtained by any one of the methods described in <1> to <3> in paraffin; and preparing a specimen from the paraffin-embedded block. <5> A centrifuge tube for preparing an embedding material, having a recess formed of agarose gel inside. <6> A kit for preparing an embedding material, comprising a centrifuge tube having a recess formed of agarose gel inside, and an agarose gel that melts upon heating. <7> The kit for preparing an embedding material according to <6>, further comprising a pipette tip for adding the agarose gel melted upon heating to the centrifuge tube having a recess formed of agarose gel inside. <8> The kit for preparing an embedding material according to <6> or <7>, wherein the agarose gel that melts upon heating is contained in a container.

[0009] The present invention provides a method for preparing an embedded material, which allows for the simple production of an embedded material without cell loss by embedding target cells or the like in a capsule made of agarose. Furthermore, a method for preparing a specimen using the embedded material is provided. In addition, a centrifuge tube for preparing the embedded material and an embedding material preparation kit including the centrifuge tube are provided.

[0010] Figure 1 is a conceptual diagram showing the method for preparing the embedded material according to this embodiment. Figure 2 shows the embedded material prepared in the example. Figure 3 shows the results of HE staining and Ki-67 staining of the specimen prepared in the example.

[0011] [Method for preparing an embedded object] The method for preparing an embedded object according to this embodiment is a method for preparing an embedded object of a target object, and comprises the following steps 1 to 6 in this order. Step 1: A centrifuge tube having a recess formed of agarose gel inside. Step 2: A suspension containing the object to be embedded is added to the centrifuge tube. Step 3: The suspension in the centrifuge tube is centrifuged to allow the object to be embedded to settle at the bottom of the recess formed of agarose gel. Step 4: The supernatant is removed. Step 5: After adding agarose gel melted by heating to the centrifuge tube, the agarose gel is cooled and rehardened, and Step 6: The embedded object in which the target object is embedded in agarose gel is removed from the centrifuge tube. According to the present invention, a method for preparing an embedded object is provided in which an embedded object can be easily obtained without loss of cells by embedding the target cells, etc. (target object) in a capsule made of agarose gel.

[0012] In this embodiment, a centrifuge tube having a recess formed of agarose gel inside is prepared, a suspension containing the material to be embedded is added to the centrifuge tube and centrifuged, the supernatant is removed and the material to be embedded is present at the bottom of the recess formed of agarose gel, after which molten agarose gel is added, the agarose gel is cooled and rehardened, and then removed to obtain an embedded material in which the material is embedded in agarose gel. With the above method, since the embedded material can be prepared with the material present in the recess formed of agarose gel, cell loss can be suppressed, and it is possible to create an embedded material while maintaining the aggregated state of the material to an extremely high degree.

[0013] The method for preparing the embedding material of this embodiment will be described with reference to Figure 1. <Step 1> As shown in Figure 1(d), Step 1 is the step of preparing a centrifuge tube 10 having a recess formed in the interior of agarose gel 22. Figure 1(d) shows two components: the centrifuge tube 10 and the agarose gel 22 in which the recess is formed.

[0014] The size and material of the centrifuge tube 10 are not particularly limited, and the volume can be any size, such as 50 mL, 15 mL, 5 mL, 2 mL, 1.5 mL, or 500 μL. The centrifuge tube 10 may be a small-volume tube with a volume of 2 mL or less, such as a microtube, and it is preferable to use a microtube when preparing a cell block.

[0015] In a first preferred embodiment of the present invention, the centrifuge tube 10 has a volume of approximately 2 mL or approximately 1.5 mL. An example of the dimensions of a microtube with a volume of approximately 2 mL or approximately 1.5 mL is as follows: Inner diameter: 9 to 11 mm, preferably 9.5 to 10.5 mm Wall thickness: 0.6 to 0.9 mm, preferably 0.65 to 0.85 mm Depth: 35 to 45 mm, preferably 37 to 43 mm. The shape of the bottom of the microtube with a volume of approximately 2 mL or approximately 1.5 mL is not limited. Examples of bottom shapes include round, conical, etc., and round is preferred. In the first preferred embodiment, the centrifuge tube 10 is preferably a microtube with a volume of approximately 2 mL and a round bottom shape.

[0016] In a second preferred embodiment of the present invention, the centrifuge tube 10 has a volume of approximately 500 μL. An example of the dimensions of a microcentrifuge tube with a volume of approximately 500 μL is as follows: Inner diameter: 6.5 to 7.5 mm, preferably 6.75 to 7.25 mm Wall thickness: 0.5 to 0.7 mm, preferably 0.55 to 0.65 mm Depth: 30 to 40 mm, preferably 33 to 37 mm. The shape of the bottom of the microcentrifuge tube with a volume of approximately 500 μL is not limited. Examples of bottom shapes include round, conical, etc., and round is preferred.

[0017] Although not shown in Figure 1, it is preferable that the centrifuge tube 10 has a lid to prevent contamination by foreign matter and to prevent leakage of the contents. Furthermore, the material of the centrifuge tube 10 is not particularly limited. From the viewpoint of adding molten agarose gel 20 to the centrifuge tube 10 to form recesses, the material of the centrifuge tube 10 needs to have heat resistance to the molten agarose gel 20, and examples include polypropylene, polystyrene, polyethylene, etc.

[0018] The centrifuge tube 10 prepared in step 1 has a recess formed of agarose gel 22 inside. The shape of the recess can be appropriately selected depending on the centrifuge tube used, the amount of suspension to be added, the amount of material to be embedded, etc., and is not particularly limited. Preferably, the tip of the recess is conical. A conical tip makes it possible to collect the material more efficiently. The dimensions of the recess are not particularly limited. In the first preferred embodiment of the present invention, when the centrifuge tube 10 is a microtube with a content volume of approximately 2 mL or approximately 1.5 mL, the dimensions of the recess formed of agarose gel are, for example, as follows: Inner diameter of opening: 5.5 to 7.5 mm, preferably 6 to 7 mm Wall thickness: 0.5 to 1.2 mm, preferably 0.75 to 1.1 mm Depth: 20 to 30 mm, preferably 23 to 28 mm Content volume: 200 to 500 μL, preferably 250 to 450 μL. Note that "wall thickness" refers to the thickness of the thinnest part of the agarose gel that forms the recess. "Depth" refers to the length from the opening formed by the agarose gel to the tip of the recess.

[0019] In a second preferred embodiment of the present invention, when the centrifuge tube 10 is a microtube with a content volume of approximately 500 μL, the dimensions of the recess formed by the agarose gel are, for example, as follows: Inner diameter of the opening: 5.0 to 5.5 mm, preferably 5.1 to 5.4 mm Wall thickness: 1.0 to 1.8 mm, preferably 1.2 to 1.6 mm Depth: 12 to 14 mm, preferably 12.5 to 13.5 mm Content volume: 150 to 250 μL, preferably 175 to 225 μL.

[0020] The method for producing a centrifuge tube having a recess formed by agarose gel 22 inside is not particularly limited. For example, as shown in Figures 1(a) and (b), a preferred method is to add molten agarose gel 20 to a centrifuge tube 10, as shown in Figure 1(c), to insert a recess-forming mold 21, for example, having a conical tip, into the molten agarose gel 20 in the centrifuge tube 10, and then, with the recess-forming mold 21 inserted, to cool the molten agarose gel to harden it into agarose gel 22, after which the recess-forming mold is removed to obtain a centrifuge tube having a recess formed by agarose gel inside. It should be noted that the recess may also be formed by cutting the agarose gel after adding molten agarose gel 20 to the centrifuge tube and cooling it to harden it, or by inserting a heated recess-forming mold 21 to form the recess.

[0021] When using the recess-forming mold 21, the shape of the recess-forming mold 21 is not particularly limited, but it is preferable that the tip of the formed recess (towards the bottom of the centrifuge tube) is conical.

[0022] In a first preferred embodiment of the present invention, when the centrifuge tube 10 is a microtube with a volume of approximately 2 mL or approximately 1.5 mL, a mold 21 for forming a recess can be made using a microtube with a volume of approximately 500 μL and a conical bottom as a mold. In a second preferred embodiment of the present invention, when the centrifuge tube 10 is a microtube with a volume of approximately 500 μL, a mold 21 for forming a recess can be made using a microtube with a volume of approximately 200 μL and a conical bottom as a mold.

[0023] Furthermore, the material of the recess-forming mold 21 is not particularly limited and should have heat resistance that is not altered by the molten agarose 20. Specifically, examples of resins include polycarbonate resin, silicone resin, phenolic resin, polyethylene terephthalate, and epoxy resin. As shown in Figure 1(c), the recess-forming mold 21 may have an alignment portion that contacts the inside of the centrifuge tube so that when inserted into the centrifuge tube 10, the recess is formed approximately in the center of the centrifuge tube when viewed from the opening of the centrifuge tube. By providing this alignment portion, the recess-forming mold 21 can be accurately held in the center of the centrifuge tube 10, making it possible to precisely form the agarose frame. In addition, by the alignment portion contacting the inside of the centrifuge tube, the recess-forming mold 21 may be fixed at a desired height when inserted into the centrifuge tube 10. Furthermore, although not explicitly shown in Figure 1(c), the locking portion may be wider than the inlet of the centrifuge tube so that it can be fixed at a desired height when inserted into the centrifuge tube 10.

[0024] <Step 2> As shown in Figure 1(e), Step 2 is the step of adding a suspension 30 containing the material to be embedded to a centrifuge tube 10 having a recess formed in the interior of agarose gel 22. The amount of suspension to be added can be adjusted as appropriate depending on the concentration of the material to be embedded in the suspension, the amount of material to be embedded required to produce a cell block, the required size of the cell block, etc. In Figure 1(e), the entire amount of suspension 30 is present in the recess, but this is not limited to this, and the suspension 30 may be added beyond the recess. However, it is preferable that the amount of suspension 30 added does not exceed the amount of the recess, as there is a risk that the suspension 30 may get between the agarose gel 22 forming the recess and the centrifuge tube 10 and cause the agarose gel 22 to peel off. In Step 3, which will be described later, it is sufficient that the settled material to be embedded 32 is present in the recess.

[0025] The embedding object is preferably at least one selected from fixed cells and fixed tissue fragments, and more preferably fixed cells or fixed tissue fragments. Here, the method of fixing cells or tissue fragments or cell lines obtained from body fluids, puncture fluids, cytological specimens, etc., is not particularly limited, but it is preferably fixed with a fixative, and examples of the fixative (fixation reagent) include aldehydes such as formaldehyde and glutaraldehyde; alcohols such as methanol and ethanol; dimethyl sveluiminate; picric acid, etc. Among these, formalin fixation using formaldehyde as the fixative is preferred from the viewpoint of high versatility, less tissue shrinkage when used with tissue fragments, and low cost. Furthermore, from the viewpoint of suitable use for the preparation of cell blocks, the embedding object in this embodiment is preferably fixed cells, and more preferably formalin-fixed cells.

[0026] <Step 3> As shown in Figure 1(f), Step 3 is a step in which the suspension 30 in the centrifuge tube 10 is centrifuged to settle the object to be embedded 32 at the bottom of the recess formed by the agarose gel 22. Step 3 separates the suspension added to the centrifuge tube in Step 2 into supernatant 34 and the object to be embedded 32. From the viewpoint of settling the object to be embedded at the bottom of the recess and suppressing the collapse of the agarose gel having the recess, it is preferable to perform the centrifugation in Step 3 using a swing rotor type centrifuge. When a fixed-angle rotor type centrifuge is used, the bottom surface of the centrifuge tube does not coincide with the direction of the centrifugal force, so the recess formed by the agarose gel may collapse, and some of the object may remain on the side of the recess, making it difficult to remove the supernatant in Step 4, which will be described later.

[0027] <Step 4> As shown in Figure 1(g), step 4 is the step of removing the supernatant 34. The removal of the supernatant may be done using an aspiration device or a micropipette, and is not particularly limited.

[0028] <Step 5> As shown in Figures 1(h) and 1(i), Step 5 is a step in which the agarose gel 40, which has been melted by heating, is added to a centrifuge tube, and then the agarose gel is cooled and rehardened. In Figure 1(h), the melted agarose gel 40 is added to the centrifuge tube 10, and in Figure 1(i), the object 32 embedded in the agarose gel 50 is formed by cooling and rehardening the melted agarose gel.

[0029] Upon re-hardening, the added agarose gel integrates with the agarose gel having pre-formed recesses in the centrifuge tube, and the object that settles at the bottom of the recess is embedded in the agarose gel. It is preferable that the molten agarose gel has the same composition as the molten agarose gel 20 added to the centrifuge tube 10 in Figure 1(b) described above. This is preferable because it produces an embedded object 32 embedded in a uniformly composed, integrated agarose gel 50.

[0030] <Step 6> As shown in Figure 1(i), Step 6 is the step of removing the embedded object, in which the object is embedded in agarose gel, from the centrifuge tube. The method of removing the embedded object from the centrifuge tube is not particularly limited, and by cutting off the tip (bottom) of the centrifuge tube and allowing air to enter the centrifuge tube from the bottom, the embedded object can be easily removed from the top of the centrifuge tube. Therefore, it is preferable to thin the thickness of the tip of the centrifuge tube by, for example, about 0.1 to 0.2 mm in advance, or to mark the part to be cut off, in order to make it easier to cut off the tip of the centrifuge tube. It is also preferable to pre-apply a thinned cut-off portion to the marked part. It is preferable that the part to be cut off is a part in which only agarose gel exists inside, so as not to cut off the object. It is also preferable that there are no protrusions (for example, constrictions, etc.) inside the centrifuge tube from the bottom to the top end so that the embedded object can be easily removed. It is also preferable that the centrifuge tube is not a self-supporting type with a skirt portion for standing upright, so that the tip of the centrifuge tube can be easily cut off.

[0031] [Method for preparing specimens] The embedded material obtained as described above is preferably made into a specimen. The method for preparing specimens preferably includes the steps of: embedding the embedded material obtained by the method for preparing embedded material of this embodiment in paraffin to prepare a paraffin-embedded block, and preparing a specimen from the paraffin-embedded block. As for the step of embedding the embedded material in paraffin to prepare a paraffin-embedded block, any known paraffin embedding method may be used as appropriate, and specifically, it is preferable to have a dehydration step, an intermediate agent treatment step, and a paraffin infiltration step in this order. The dehydration step is preferably a step of replacing water with ethanol, and if necessary, it is preferable to treat it multiple times with ethanol diluted with water in stages and then transfer it to high-concentration ethanol to replace the water with ethanol. However, it is not limited to the above embodiment, and since the object is already fixed, it may be treated multiple times with 100% ethanol. The intermediate agent treatment step is a step of removing ethanol by treating it multiple times with an intermediate agent such as xylene or chloroform, since paraffin does not mix with ethanol. The paraffin impregnation process involves placing the embedding material into paraffin melted by heating and then cooling and solidifying it. Alternatively, the embedding material may be placed in the paraffin multiple times after being treated with melted paraffin, and then cooled and solidified. This process yields a paraffin-embedded block.

[0032] The process of preparing a specimen from a paraffin-embedded block involves slicing the paraffin-embedded block into thin sections (specimens). Section preparation can be carried out according to known methods, for example, using a microtome. The sections are preferably placed on a glass slide, in which case they may be floated in a water bath to spread before being placed on the glass slide and then dried.

[0033] The specimen may be stained or otherwise treated using conventional methods, depending on the intended observation or examination. Specific examples include HE staining, fat staining, or immunohistochemical staining methods such as enzyme-mediated immunohistochemistry (DAB) or immunofluorescence.

[0034] [Centrifuge tube and kit for preparing embedded material] This embodiment also discloses a centrifuge tube and a kit for preparing embedded material. The centrifuge tube for preparing embedded material in this embodiment is a centrifuge tube having a recess formed of agarose gel inside, and is the same as the centrifuge tube having a recess formed of agarose gel inside prepared in step 1 of the method for preparing embedded material described above, and the preferred range is also the same.

[0035] Furthermore, the embedding kit of this embodiment includes a centrifuge tube having a recess formed of agarose gel inside, and an agarose gel that melts upon heating. In addition to the centrifuge tube and the agarose gel that melts upon heating described above, the embedding kit may also preferably include a pipette tip for adding the agarose gel that has melted upon heating to the centrifuge tube. The agarose gel that melts upon heating is preferably contained in a container, and it is preferable that the necessary amount of agarose gel, depending on the volume that can be added to the centrifuge tube having a recess formed of agarose gel inside, is contained in a container that can be heated to a temperature above the melting temperature of the agarose gel. The agarose gel that melts upon heating is preferably the same composition as the agarose gel that forms the recess inside the centrifuge tube. Furthermore, because the melted agarose gel is viscous, it is difficult to aspirate and dispense it with a regular micropipette tip. Therefore, a pipette tip with a wide opening is preferable. For example, the tip of a pipette tip may be cut off to widen the opening.

[0036] [Applications] The embedded material obtained by the embedding method of this embodiment is suitably used for the preparation of cell blocks by embedding it in paraffin. By using the cell block, thin sections of cell specimens can be prepared in the same way as tissue specimens, and it can be used not only for IHC and ISH methods but also for gene-related tests, making it extremely useful as a pathological specimen. Furthermore, the centrifuge tube and embedding kit for embedding of this embodiment are suitably used for the preparation of embedding for the preparation of cell blocks.

[0037] 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.

[0038] <Step 1: Preparation of a centrifuge tube with a recess formed by agarose gel inside> 3 g of agarose (Agarose S (product code: 312-01193, 100 g), manufactured by Nippon Gene Co., Ltd.) was added to 100 mL of phosphate-buffered saline (PBS), and dissolved by boiling at 100°C to prepare a 3% agarose solution. Next, 1 mL of the above-mentioned 3% agarose solution was added to a 2.0 mL centrifuge tube (Biolamo Microtube Round Bottom 2 mL, product code: 1-1600-02). Since the 3% agarose solution is viscous, the tip of a micropipette (Pipetteman P1000, manufactured by Gilson) was cut off and used. A spindle-shaped mold (made of epoxy resin) was inserted into the molten agarose gel, and the mixture was left to stand at room temperature to solidify (re-harden) the agarose, obtaining an agarose gel with a recess. After the agarose gel had solidified, the mold was removed, and a centrifuge tube was obtained with a spindle-shaped recess formed by the agarose gel inside.

[0039] <Step 2: Adding the suspension containing the embedding material to the centrifuge tube> Cells for which a cell block was to be prepared (in this example, the colon cancer cell line HCT-116 was used) were fixed with 10% neutral buffered formalin for about 12 hours. After fixation, the cells were washed twice with PBS. The fixed cells were stirred with about 300 μL of PBS to prepare a cell suspension. The cell suspension was placed in a centrifuge tube having a recess formed with 3% agarose gel.

[0040] <Step 3: Centrifugation of the suspension in the centrifuge tube to settle the embedding material at the bottom of the depressions formed by the agarose gel> The centrifuge tube to which the cell suspension was added was centrifuged in a swing-rotor type centrifuge at 1,000 rpm (approximately 180-200 g, 5 minutes) to settle the cells at the bottom of the depressions formed by the agarose gel and form a cell pellet.

[0041] <Step 4: Step to remove supernatant> After centrifugation, the cells are separated into a pellet and a supernatant (PBS). The supernatant was discarded, leaving only the pellet.

[0042] <Step 5: Adding agarose gel melted by heating to a centrifuge tube, then cooling and re-hardening the agarose gel> A 3% agarose gel, stored in a 1.5 mL screw-cap tube (product code: 63-2980-67, screw-cap microtube 1.5 mL), was prepared in advance by melting it in a 100°C heat block. The 3% agarose gel melted at 100°C was aspirated using a micropipette (Pipetteman P1000, Gilson) fitted with a cut tip and injected into a centrifuge tube where the cell pellet was located at the bottom of the recess. Due to the heat of the melted agarose gel, the added melted agarose gel welded to the agarose inside the centrifuge tube, and re-hardened while incorporating the settled cell pellet.

[0043] <Step 6: Step to remove the embedded object from the centrifuge tube> The tip of the 2.0 mL centrifuge tube was cut off with a cutter to a width of about 5 mm, and the embedded cell pellet embedded in agarose gel was removed from the mouth of the tube. Excess agarose was cut off, and the embedded object with the cell pellet was embedded in paraffin as shown below to prepare a paraffin-embedded block. Figure 2(a) shows the obtained embedded cell material embedded in agarose gel. As shown in Figure 2(b), the unnecessary agarose gel portion of the embedded object in which the cells are embedded in agarose gel was trimmed (removed), and as shown in Figure 2(c), it was embedded in paraffin in the fourth chamber so that the tip of the centrifuge tube was the thin section surface.

[0044] <Preparation of paraffin-embedded blocks> Using a closed automatic fixation and embedding device (Tissue-Tek (registered trademark) VIP 6 AI, Sakura Finetek Japan Co., Ltd.), dehydration steps (100% alcohol (solvent ethanol 100 for pathological staining, Mutoh Chemical Co., Ltd., product number: 43105), 7 tanks), intermediate agent treatment steps (xylene (xylene, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., product number: 249-00097), 3 tanks), and paraffin infiltration steps (Paraplast 60GR, Mutoh Chemical Co., Ltd., product number: 43257, 4 tanks) were performed to infiltrate each chemical solution into the embedding material. The immersion time in each tank was as follows. Alcohol: 7 tanks (1 hour each), Xylene: 2 tanks (1 hour each), Xylene: 1 tank (1 hour 30 minutes), Paraffin: 4 tanks (63°C, 1 hour 30 minutes each). In the fourth tank of the paraffin layer, the embedding material in which the cells were embedded in the agarose gel was placed in a mold (embedding dish), and then the embedding dish was placed on a cooling table at 0°C or lower to rapidly solidify the paraffin, obtaining a paraffin-embedded block.

[0045] [Preparation of specimens] The obtained paraffin-embedded block was thinly sliced to a thickness of 3 μm using a microtome. The obtained sections were stretched in a water bath, placed on a slide glass, and then completely dried. On the slide glass, the sections were stained with (i) HE staining and (ii) Ki-67 antibody as immunohistochemical staining according to the conventional method and observed. The results are shown in Figure 3 below. The Ki-67 protein is a cell proliferation ability marker (proliferating cell marker) and is present in all cell cycles (G1 phase, S phase, G2 phase, M phase) of proliferating cells, but is not present in the quiescent (G0) phase, so it is used for the evaluation of proliferation ability.

[0046] As shown in Figure 3, in the specimens using the cell block, the dissipation and disappearance of cells were suppressed, and sufficient evaluation was possible.

[0047] 10: Test tube, 20: Melted agarose gel, 21: Mold for forming recess, 22: Solidified agarose gel, agarose gel with recess formed, 30: Suspension, 32: Embedding object, 34: Supernatant, 40: Melted agarose gel, 50: Integrated agarose gel, 100: Embedding material of object

Claims

1. A method for producing an embedded object, comprising the following steps 1 to 6 in this order: Step 1: Prepare a centrifuge tube having a recess formed of agarose gel inside Step 2: Add a suspension containing the object to be embedded to the centrifuge tube Step 3: Centrifuge the suspension in the centrifuge tube to allow the object to be embedded to settle at the bottom of the recess formed of agarose gel Step 4: Remove the supernatant Step 5: Add agarose gel melted by heating to the centrifuge tube, then cool and reharden the agarose gel, and Step 6: Remove the embedded object from the centrifuge tube, in which the object has been embedded in the agarose gel.

2. The method for preparing an embedding object according to claim 1, wherein the embedding object is fixed cells or fixed tissue fragments.

3. The method for preparing an embedded object according to claim 1, wherein the centrifugal separation in step 3 is performed using a swing rotor type centrifuge.

4. A method for preparing a specimen, comprising the steps of: preparing a paraffin-embedded block by embedding an embedded material obtained by the method of claim 1 in paraffin; and preparing a specimen from the paraffin-embedded block.

5. A centrifuge tube for preparing embeddings, having a recess formed of agarose gel inside.

6. A kit for preparing embeddings, comprising a centrifuge tube having a recess formed of agarose gel inside, and an agarose gel that melts upon heating.

7. The embedding kit according to claim 6, further comprising a pipette tip for adding agarose gel melted by heating to a centrifuge tube having a recess formed in the interior of agarose gel.

8. The embedding kit according to claim 6 or 7, wherein the agarose gel that melts upon heating is contained in a container.

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