Method for preparing paraffin sections

Abstract

To provide a method for manufacturing a piece which can be three-dimensionally analyzed from a paraffin embedded tissue block, and the piece manufactured by the method.SOLUTION: The method for manufacturing a paraffin piece includes a step of processing a paraffin embedded sample with a mixture solution of a paraffin melting agent and a hydrophilic agent before slicing the paraffin embedded sample. The present invention relates to the method, the paraffin piece manufactured by the method, and the mixture solution.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for preparing paraffin sections from paraffin-embedded specimens. More specifically, it relates to a method for preparing paraffin sections that enables both two-dimensional analysis and three-dimensional analysis.

Background Art

[0002] In order to visually capture a biological tissue or a pathological tissue, microscopic observation of a biological tissue specimen has been carried out for a long time. Even currently, the importance of visually observing a biological tissue remains unchanged, but in order to more accurately grasp the state of the tissue, in addition to two-dimensional observation, three-dimensional analysis of the tissue has also come to be carried out. However, until now, when performing three-dimensional tissue analysis after observing a paraffin section obtained by slicing a paraffin-embedded specimen to about 10 μm or less with an optical microscope or the like, it has been necessary to use a sample different from the tissue sample observed under the microscope. Further, as a three-dimensional analysis method, continuous photographing of paraffin sections of about 10 μm or less and construction of a three-dimensional image by data processing have been carried out, but such a method has a huge amount of work, and often the desired information cannot be obtained. Furthermore, when two-dimensional observation and three-dimensional analysis are performed on different samples, it is difficult to directly link the results of both, and it may be difficult to accurately grasp the state of the tissue. In particular, when targeting a small amount of precious tissue, it has been very difficult to prepare samples for two-dimensional observation and three-dimensional analysis separately.

[0003] In view of the above situation, there is an increasing need for paraffin sections of a thickness that enables effective three-dimensional analysis by a scanning electron microscope (SEM) or the like from paraffin-embedded specimens. Although there are some documents that have mentioned paraffin sections with a thickness of about 300 μm (Patent Document 1 and Non-Patent Document 1), there are no reports on their detailed preparation methods and practicality. Therefore, there remains a strong need to establish a practical method for preparing thick paraffin sections that allow for easy three-dimensional analysis. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2009-80108 [Non-patent literature]

[0005] [Non-Patent Document 1] Feldengut et al., J Neurosci Methods. 15:241-244 2013. [Overview of the project] [Problems that the invention aims to solve]

[0006] In view of the above circumstances, the present invention aims to provide a method for preparing sections (thicker than conventional sections) from paraffin-embedded tissue blocks that allow for three-dimensional analysis, and to provide sections prepared by this method. [Means for solving the problem]

[0007] The inventors successfully prepared sections suitable for three-dimensional analysis by applying or mounting (placing the mixed solution on) a mixed solution of a paraffin melting agent such as xylene and a hydrophilizing agent such as ethanol onto the surface of a paraffin block (the block surface parallel to the cut surface) and letting it stand for several minutes, and then preparing sections of 100 μm or larger using a microtome. When thick sections of about 100 μm thickness are prepared using conventional methods, the sections tend to fragment or curl, making three-dimensional analysis difficult. The inventors hypothesized that improving the flexibility of the specimen tissue during the deparaffinization process could avoid the above phenomena (fragmentation and curling of sections). They treated paraffin-embedded specimens with a mixed solution of a paraffin melting agent and a hydrophilizing agent, and then prepared sections with a thickness of about 50 μm to 300 μm from these specimens. Despite being thicker than 100 μm, the resulting sections retained their tissue flexibility, did not fragment or curl, and were in good condition, sufficient for subsequent analysis.

[0008] The inventions completed based on the above findings are as follows (1) to (17). (1) A method for preparing paraffin sections, comprising the step of treating with a mixed solution of a paraffin melting agent and a hydrophilizing agent. (2) The method according to (1) above, wherein the ratio of paraffin melting agent and hydrophilizing agent in the mixed solution is 1:0.5 to 1:4 by volume. (3) The method according to (1) or (2) above, wherein the paraffin melting agent is one or more selected from the group consisting of xylene, limonene, and pinene. (4) The method according to (1) or (2) above, wherein one or more are selected from the group consisting of the hydrophilizing agent, ethanol, propanol, and acetone. (5) Paraffin sections obtained by slicing a paraffin-embedded specimen that has been treated in a step comprising treatment with a mixed solution of a paraffin melting agent and a hydrophilizing agent. (6) The paraffin section according to (5) above, comprising the step of treating with a mixed solution of a paraffin melting agent and a hydrophilizing agent, followed by treatment with water. (7) A paraffin section as described in (5) or (6) above, having a thickness of 50 μm or more and 300 μm or less. (8) A pretreatment agent for preparing paraffin sections, comprising a mixed solution of a paraffin melting agent and a hydrophilizing agent. (9) The pretreatment agent according to (8) above, wherein the ratio of the paraffin melting agent and the hydrophilizing agent in the mixed solution is 1:0.5 to 1:4 by volume. (10) The pretreatment agent according to (8) or (9) above, wherein the paraffin melting agent is one or more selected from the group consisting of xylene, limonene, and pinene. (11) The pretreatment agent according to (8) or (9) above, which is one or more selected from the group consisting of the hydrophilizing agent, ethanol, propanol, and acetone. (12) A method for analyzing a paraffin-embedded specimen, comprising preparing paraffin sections from a paraffin-embedded specimen treated with a mixed solution of a paraffin melting agent and a hydrophilizing agent, and analyzing the paraffin sections in three dimensions. (13) The analytical method according to (12) above, wherein the ratio of paraffin melting agent and hydrophilizing agent in the mixed solution is 1:0.5 to 1:4 by volume. (14) The analytical method according to (12) or (13) above, wherein the paraffin melting agent is one or more selected from the group consisting of xylene, limonene, and pinene. (15) The analytical method according to (12) or (13) above, wherein one or more of the group consisting of the hydrophilizing agent, ethanol, propanol, and acetone are selected. (16) Analysis image obtained using the analysis method described in (12) above. (17) The analysis image described in (16) above, with a depth of field of 2 μm to 100 μm. In this specification, the symbol "~" indicates a numerical range that includes the values ​​to its left and right. [Effects of the Invention]

[0009] Sections (with a thickness of approximately 50 μm or more) prepared by the method of the present invention are good sections that can be used for various analyses, including three-dimensional analysis. Therefore, the present invention makes it possible to perform both two-dimensional and three-dimensional analysis using the same paraffin-embedded specimen, and to collect more information than before. [Brief explanation of the drawing]

[0010] [Figure 1] The left section of FIG. 1 is a section prepared from a paraffin-embedded specimen pretreated with a mixed solution of xylene and ethanol, which is a pretreatment according to an embodiment of the present invention, and the right section is a section prepared from a paraffin-embedded specimen without performing the pretreatment according to the embodiment of the present invention. [Figure 2] Sections prepared from paraffin-embedded specimens pretreated with various mixed solutions. The arrow indicates a section deformed by drying. Xy indicates xylene, and Et indicates ethanol. [Figure 3] Stereomicroscopic image of the sliced surface of the section. [Figure 4] Sections sliced after heat treatment (100 μm thick). Photographs of the sectioned specimen (Xy+Et) and the heat-treated sections (50°C 10 min, 50°C 15 min, 60°C 10 min) are shown after pretreating the paraffin-embedded specimen with xylene and ethanol. [Figure 5] Sections sliced after heat treatment (300 μm thick). Photographs of the sectioned specimen (Xy+Et) and the heat-treated sections (60°C 5 min, 60°C 5 min, 60°C 10 min) are shown after pretreating the paraffin-embedded specimen with xylene and ethanol. The arrow indicates the part where the tissue has fallen off from the paraffin block. [Figure 6] Photograph comparing the thickness of the sections. Upper tissue: 100 μm section, lower tissue: 300 μm section. [Figure 7] Observation images of the cilia of the ependymal cells. A: HE staining of the ependymal cells of the ventricle, B: SEM image of the consecutive tissue of A, C: Enlarged image of B. [Figure 8] Observation images of the choroid plexus. A: HE staining of the choroid plexus, B: SEM image of the consecutive tissue of A. [Figure 9] Observation images of the epithelium of the nasal cavity. A: HE staining of the nasal cavity, B: Enlarged image (broken line) within A (arrow (black) indicates olfactory epithelium, arrow (hollow) indicates respiratory epithelium), C: SEM image of the consecutive tissue of B, and each arrow indicates the same tissue as in B, D: SEM image of the respiratory epithelium within the frame (solid line) of A. [Figure 10]Observation images of hair cells in the Corti器. A: HE staining of the cochlea, B: SEM image of a serial tissue of A, C: Enlarged image of the area within the frame (dashed line) in B, D: Enlarged image of the area within the frame (solid line) in B. [Figure 11] Observation images of alveolar epithelial cells. A: HE staining of alveoli, B: SEM image of a serial tissue of A. [Figure 12] Observation images of glomeruli. A: SEM image of glomeruli (arrow indicates glomerulus), B: Podocytes of glomeruli. [Figure 13] Three-dimensional analysis of alveoli by fluorescence immunostaining. Blue: each stain, Green: HOPX (immature type I alveolar epithelium), Magenta: surfactant protein C (type II alveolar epithelial cells)

Mode for Carrying Out the Invention

[0011] Hereinafter, the mode for carrying out the present invention will be described. The first embodiment is a method for preparing a paraffin section, which includes a step of treating a paraffin-embedded specimen with a mixed solution of a paraffin melting agent and a hydrophilic agent (hereinafter also referred to as "the pretreatment mixed solution in the present embodiment"). Further, the first embodiment also includes paraffin sections obtained by thinly slicing the paraffin-embedded specimen treated with the mixed solution of the paraffin melting agent and the hydrophilic agent. A paraffin-embedded specimen is a specimen in which paraffin has penetrated into an organ or tissue, and is widely used for morphological observation of tissues and the like. The paraffin-embedded specimen in the present embodiment may be any method for its preparation as long as it is a tissue specimen penetrated with paraffin. Also, the tissue or the like to be targeted may be any one. The method according to this embodiment is characterized by including a step of treating the portion of the paraffin-embedded specimen that will be sectioned (the portion that will become a section after slicing) with a mixed solution of a paraffin threshing agent and a hydrophilizing agent before slicing the paraffin-embedded specimen (hereinafter also referred to as the "pretreatment step according to this embodiment"). Furthermore, in order to maintain appropriate moisture retention and to make it easier to slice sections of any desired thickness, the "pretreatment step according to this embodiment" also includes a step of treating with water after treating with the mixed solution of the paraffin threshing agent and the hydrophilizing agent, which is another characteristic of the method according to this embodiment. The step of treating with water can be optionally performed depending on the condition of the paraffin-embedded specimen. In this way, by performing the pretreatment according to this embodiment, the flexibility of the tissue contained in the sliced ​​section is improved, and it becomes possible to prevent deformation such as fragmentation and curling of the section. Therefore, paraffin sections prepared by the method according to this embodiment have structural differences from sections obtained by conventional methods, i.e., sections obtained by slicing paraffin-embedded specimens without pretreatment with a mixed solution of paraffin-thawing agent and hydrophilizing agent (especially sections of 50 μm or more or 100 μm or more), in that they have improved tissue flexibility and do not fragment or curl.

[0012] The paraffin melting agent according to this embodiment is not particularly limited as long as it is an organic solvent that can dissolve paraffin. Examples include aromatic hydrocarbons such as toluene and xylene (o-xylene, m-xylene, p-xylene or mixtures thereof), aliphatic hydrocarbons such as n-hexane, cyclohexane, and petroleum ether, and essential oils such as limonene and pinene (α-pinene, β-pinene or mixtures thereof). These may be used individually or in mixtures of two or more. Preferably, it is a volatile organic solvent, such as xylene, pinene, or limonene, and more preferably xylene. Furthermore, the hydrophilizing agent is an organic solvent that can be dissolved in paraffin solvent and also in water, and is not particularly limited as long as it has the effect of retaining an appropriate amount of moisture in the excised tissue section. Examples include alcohol compounds such as ethanol and propanol (1-propanol or 2-propanol), polyhydric alcohol compounds such as 1,2-ethanediol, 1,3-propanediol, 1,4-butanediol, and glycerol, and ketone compounds such as acetone and diethyl ketone. These may be used individually or in mixtures of two or more. Preferably, ethanol, acetone, and 2-propanol are used, and ethanol is more preferred.

[0013] The mixing ratio of the paraffin melting agent and the hydrophilizing agent constituting the pretreatment mixture in this embodiment is not particularly limited as long as it dissolves the paraffin and the paraffin melting agent and the hydrophilizing agent form a uniform solution as a mixture. For example, the volume ratio of paraffin melting agent to hydrophilizing agent is preferably 1:0.5 to 1:5, more preferably 1:0.5 to 1:4, more preferably 1:0.5 to 1:2, and most preferably 1:1.

[0014] The pretreatment method for paraffin-embedded specimens can be any method that ensures the mixed solution according to this embodiment permeates uniformly into the portion to be sectioned. For example, an appropriate amount of the mixed solution according to this embodiment may be applied to the surface of the portion to be sectioned (for example, the surface that will have the largest surface area when sectioned), or the mixed solution may be applied and then left to stand for a few minutes until the mixed solution permeates into the portion to be sectioned. After removing the mixed solution, treatment with water may be performed as needed. After pre-treating the paraffin-embedded sections with the mixture according to this embodiment, sections of the desired thickness can be prepared using a microtome or the like according to a conventional method. The obtained sections can then be deparaffinized and used for tissue staining and the like.

[0015] The paraffin-embedded specimens pre-treated according to this embodiment, even when sliced ​​to a thickness of 30 μm or more, for example, 100 μm or more or 200 μm or more, the resulting sections are of sufficient quality to be used not only for two-dimensional analysis but also for three-dimensional analysis, such as obtaining continuous images with a confocal microscope, without deformation such as fragmentation or curling. Images obtained through two-dimensional analysis are images that contain information in two dimensions, the X and Y axes, while images obtained through three-dimensional analysis are images that contain information in the Z axis direction in addition to the X and Y axes. The depth in the Z axis direction (hereinafter also referred to as "depth of field") of the image obtained through three-dimensional analysis according to the present invention varies depending on the three-dimensional analysis method, but is between 1 μm and 200 μm, and preferably between 10 μm and 150 μm. The sliced ​​section in this embodiment has a thickness of 30 μm or more, and there is no upper limit to the thickness as long as it does not hinder two-dimensional or three-dimensional analysis, but it is preferably 50 μm to 300 μm thick, more preferably 50 μm to 200 μm thick, and most preferably 50 μm to 150 μm thick. Furthermore, since the optimal thickness differs depending on the method used for three-dimensional analysis, it is necessary to select the section thickness in this embodiment according to the method used for three-dimensional analysis.

[0016] The second embodiment is a pretreatment agent for preparing paraffin sections, which consists of a mixed solution of a paraffin melting agent and a hydrophilizing agent (hereinafter also referred to as "the treatment agent according to this embodiment"). The treatment agent according to this embodiment is used to treat paraffin-embedded specimens before slicing them in order to prevent deformation of sections when preparing sections of approximately 30 μm or larger from paraffin-embedded specimens. The treatment agent consists of a mixed solution of a paraffin-thawing agent and a hydrophilizing agent. For details on the paraffin-thawing agent, the hydrophilizing agent, and their mixing ratio, please refer to the description in the first embodiment.

[0017] The third embodiment is a method for analyzing a paraffin-embedded specimen, which includes preparing paraffin sections from a paraffin-embedded specimen treated with a mixed solution of a paraffin melting agent and a hydrophilizing agent, and analyzing the paraffin sections in three dimensions. Previously, when analyzing tissue samples in three dimensions, hundreds of serial thin sections, each only a few micrometers thick, were prepared, and the microscopic images of each section were processed to construct a three-dimensional image. This method required an enormous amount of work, and the resulting images did not always achieve the desired objective. According to the paraffin section preparation method of this embodiment, sections with a thickness of 30 μm or more, and even 100 μm or more, can be prepared. The prepared sections can be subjected to three-dimensional analysis using a scanning electron microscope (SEM), a confocal laser microscope, or Golgi staining with an optical microscope. Therefore, the analysis method of this embodiment involves cutting out sections for conventional two-dimensional analysis (thickness of several μm) from a single paraffin-embedded specimen, processing the cut surface with the pretreatment step of this embodiment, and cutting out sections that can be used for three-dimensional analysis. As a result, sections for both two-dimensional and three-dimensional analysis can be prepared from a single paraffin-embedded specimen, and three-dimensional information with continuity with two-dimensional information can be obtained, allowing for the collection of more accurate tissue information. Furthermore, an additional feature of the three-dimensional analysis in this embodiment is that the cut lumen (internal space of a tubular organ) can be observed from the side, and images of the surface structure of cells facing the lumen can be obtained. Specifically, by cutting a paraffin section of appropriate thickness (for example, a section with a thickness of 100 μm) obtained by the method according to this embodiment, and performing a three-dimensional analysis of the cut surface from the side, the desired image can be easily obtained.

[0018] Where this specification is translated into English and contains the singular forms of "a," "an," and "the," it shall be understood to include both singular and plural forms unless the context clearly indicates otherwise. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Examples]

[0019] 1. Materials and Methods 1-1. Preparation of a mixed solution of paraffin melting agent and hydrophilizing agent The above mixed solution was prepared by mixing equal amounts of xylene (pathology-grade xylene MS; Mutoh Chemical Co., Ltd.) and ethyl alcohol (pathology-grade staining solvent ethanol 100; Mutoh Chemical Co., Ltd.). In addition, as alternative solvents for xylene, equal-volume mixtures of D(+)-limonene (Remosol®; Fujifilm Wako Pure Chemical Corporation) and pinene compounds (Remosol Ace; Fujifilm Wako Pure Chemical Corporation) with ethyl alcohol (pathology-grade staining solvent ethanol 100; Mutoh Chemical Co., Ltd.) were prepared. Furthermore, equal-volume mixtures of xylene (pathology-grade xylene MS; Mutoh Chemical Co., Ltd.) and acetone (Nacalai Tesque Co., Ltd.) and isopropanol (2-propanol; Nacalai Tesque Co., Ltd.) were prepared as alternative solvents for ethanol.

[0020] 1-2. Collection of paraffin sections A sliding microtome (IVS-410; Sakura FineTech Japan Co., Ltd.) was used to collect paraffin sections. The paraffin blocks used for section collection were obtained by perfusion fixing of various organs from rats and mice with 4% paraformaldehyde (paraformaldehyde; Fujifilm Wako Pure Chemical Corporation), fixing them with 10% neutral buffered formalin (formalin stock solution; Mutoh Chemical, phosphate-buffered physiological saline powder; Fujifilm Wako Pure Chemical Corporation), and then embedding them in paraffin (Tissue-Tec® Paraffin Wax II 60; Sakura FineTech Japan Co., Ltd.) according to standard procedures. For thin sectioning, the paraffin block was trimmed to the target area, and the mixed solution prepared in 1-1 above was placed on the thin section surface of the paraffin block and left to stand for 2-3 minutes. After that, the solution was absorbed with tissue paper, water was added, and after standing for 2-3 minutes, the water was absorbed and removed with tissue paper. Next, sections 100 μm or 300 μm thick were collected using a microtome. Furthermore, because tissues with multiple organs or high cell density tend to detach from paraffin during sectioning, cellophane tape (registered trademark) was applied to the surface of the section before collection.

[0021] 1-3. Comparison with heat treatment For heat-treated sectioning, the section surface was heated on a hot plate (Hot plate HTP452AB; ADVANTEC Toyo Co., Ltd.) at 50°C for 10 or 15 minutes, or at 60°C for 5 or 10 minutes, and then immediately sectioned using a microtome set to a section thickness of 100 μm or 300 μm.

[0022] 1-4. SEM (Scanning Electron Microscope) Observation 100 μm sections of mouse and rat brain, nasal cavity, cochlea, kidney, and lung were deparaffinized according to standard procedures, washed with water, and re-fixed for 30 minutes in a 2.5% glutaraldehyde aqueous solution (glutaraldehyde; Nacalai Tesque Co., Ltd.). After washing with water, the sections were electrically treated for 30 minutes in a 2.5% phosphotungstic acid aqueous solution (12-tungst(VI) phosphate n-hydrate), and then gradually dehydrated with 2.5% ethyl alcohol phosphotungstate. The opposite side of the tissue to be observed was attached to double-sided carbon tape for SEM (Nisshin EM Co., Ltd.), and dried using a simple vacuum device (vacuum pan case; Skater Co., Ltd.). The dried samples were observed for tissue surface structure using a low-vacuum scanning microscope (TM3030; Hitachi, Ltd.). Sections for SEM observation were taken from continuous sections of sections previously used for normal HE staining.

[0023] 1-5. Three-Dimensional Structure of the Organization To analyze the distribution of alveolar epithelial cells in three dimensions, mouse lung tissue was used to acquire sequential images at 1 μm intervals using a confocal laser microscope, and a three-dimensional image was constructed. For immunofluorescence staining, 100 μm thick sections of mouse lung (sections prepared according to the method of this embodiment) were deparaffinized according to a conventional method, washed with water, and blocked with 1% BSA (bovine serum albumin; Sigma-Aldrich) / 0.01 M PBS for 1 day. Primary antibodies (anti-HOPX antibody; Santacruz co. Ltd., anti-Surfactant protein C antibody; Santacruz co. Ltd.) and secondary antibodies were diluted with blocking reagents and reacted for 3 days. Observation was performed using a confocal laser microscope (Leica TCS SP5 II), and 3D reconstruction was performed using image analysis software (imageJ).

[0024] 2.Results 2-1. Investigation of the effects of a mixed solution of paraffin melting agent and hydrophilizing agent To obtain thick sections from paraffin blocks, the effectiveness of various solvents in sectioning was evaluated. As a result, good thin sections were obtained with all combinations (Table 1, Figure 1 (left section) and Figure 2). When sections were prepared without pretreatment with a mixed solution of paraffin solvent and hydrophilizing agent, the sections became fragmented (Figure 1, (right section: without treatment with a mixture of xylene and ethanol)). [Table 1]

[0025] Mixtures of ethanol and various paraffin-melting solvents yielded good sections in all combinations (Table 2, Figure 2). However, paraffin-melting agents alone (without ethanol treatment) could not withstand shrinkage during drying, resulting in significant deformation (Table 2, Figure 2). [Table 2]

[0026] Sections prepared by pretreatment with a mixed solution of paraffin melting agent and hydrophilizing agent were compared with sections prepared by hydrophilic treatment alone (Table 3, Figure 3). As a result, it was found that fine cracks occurred in the sections when hydrophilic treatment alone was performed, indicating that in order to obtain thicker sections (e.g., 50 μm or more), it is necessary to treat the sections with the mixed solution of paraffin melting agent and hydrophilizing agent according to the present invention before thin slicing (Table 3, Figure 3). [Table 3]

[0027] 2-2. Comparison of pretreatment with a mixed solution of paraffin melting agent and hydrophilizing agent and heat treatment Since a thin sectioning method utilizing the softening of paraffin by heat has been reported in the past (Non-Patent Literature 1), a comparative study was conducted with the effects of the method according to the present invention. As a result, no damage such as cracks was observed in 100 μm thick sections that were thinly sliced ​​after pretreatment with the mixed solution according to the embodiment of the present invention. In contrast, it was found that with the method of thin sectioning after heat treatment, the paraffin portion other than the tissue could be thinly sliced ​​well, but cracks occurred in the tissue (Figure 4).

[0028] 2-3. Examination of the thickness of sections produced by the method including the pretreatment step according to this embodiment. To investigate the preparation of sections larger than 100 μm, 300 μm sections were prepared. As a result, the desired sections could be obtained from paraffin-embedded specimens pretreated according to this embodiment after 25 minutes of processing (Figure 6). On the other hand, with the heat treatment method, although the specimens were kept at 60°C, tissue cracking and tissue detachment from the paraffin block were observed (Figure 5).

[0029] 2-4. SEM Observation Paraffin sections (100 μm thick) were prepared using a method that included a step of pretreatment with a mixed solution of a paraffin melting agent and a hydrophilizing agent, and various observations were performed. Clear cilia of ependymal cells were observed on the lateral surfaces of the ventricles in the mouse brain (Figure 7). In the choroid tissue, microvilli and secretions of choroidal epithelial cells were clearly observed (Figure 8). In the nasal cavity tissue of mice, the boundary between the olfactory epithelium and respiratory epithelium was clearly distinguishable, and cilia and secretions of respiratory epithelial cells were also observed (Figure 9). In the cochlea of ​​mice, the outer hair cells and their arrangement were clearly observed in the organ of Corti (Figure 10). In rat lungs, an increase in alveolar macrophages was observed in HE staining, and a similar increase in alveolar macrophages was observed in SEM images of the same area. Furthermore, the alveolar walls and alveolar ducts were clearly observed in the SEM images (Figure 11). In rat kidneys, glomerular podocytes were clearly observed (Figure 12). Furthermore, these SEM-observed tissues did not show damage that would affect the observation results compared to HE staining of adjacent tissues.

[0030] 2-6. Building a Three-Dimensional Image of the Organization Observation using a confocal laser microscope allowed us to detect fluorescence up to a depth of 30 μm from the tissue surface and construct a three-dimensional image of a tissue with a thickness of 30 μm (Figure 14). [Industrial applicability]

[0031] This invention provides a method for preparing sections that are thicker (50 μm or more) than conventional paraffin sections and capable of various analyses. Therefore, this invention provides a new means for analyzing pathological tissue and is expected to be used in fields such as medicine.

Claims

1. A method for preparing paraffin sections from a paraffin-embedded specimen, comprising the step of treating the paraffin-embedded specimen with a mixed solution of a paraffin melting agent and a hydrophilizing agent before slicing.

2. The method according to claim 1, wherein the ratio of the paraffin melting agent and the hydrophilizing agent in the mixed solution is 1:0.5 to 1:4 by volume.

3. The method according to claim 1 or claim 2, wherein the paraffin melting agent is one or more selected from the group consisting of xylene, limonene, and pinene.

4. The method according to claim 1 or claim 2, wherein the hydrophilizing agent is one or more selected from the group consisting of ethanol, propanol, and acetone.

5. A method for preparing paraffin sections, characterized by thinly slicing a paraffin-embedded specimen that has been treated with a mixed solution of a paraffin melting agent and a hydrophilizing agent.

6. A method for preparing paraffin sections according to claim 5, characterized in that a paraffin-embedded specimen treated with a mixed solution of the paraffin melting agent and the hydrophilizing agent is treated with water and then sliced.

7. The method for preparing paraffin sections according to claim 5 or claim 6, characterized in that the paraffin-embedded specimen is sliced ​​into pieces of 50 μm or more and 300 μm or less.

8. A pretreatment agent used for preparing paraffin sections from a paraffin-embedded specimen, comprising a mixed solution of a paraffin-melting agent and a hydrophilizing agent, for treating the paraffin-embedded specimen before slicing it.

9. The pretreatment agent according to claim 8, wherein the ratio of the paraffin melting agent and the hydrophilizing agent in the mixed solution is 1:0.5 to 1:4 by volume.

10. The pretreatment agent according to claim 8 or 9, wherein the paraffin melting agent is one or more selected from the group consisting of xylene, limonene, and pinene.

11. The pretreatment agent according to claim 8 or 9, which is one or more selected from the group consisting of the hydrophilizing agent, ethanol, propanol, and acetone.

12. A method for analyzing paraffin-embedded specimens, The analytical method comprises preparing paraffin sections from a paraffin-embedded specimen treated with a mixed solution of a paraffin melting agent and a hydrophilizing agent, and performing a three-dimensional analysis of the paraffin sections.

13. The analytical method according to claim 12, wherein the ratio of the paraffin melting agent and the hydrophilizing agent in the mixed solution is 1:0.5 to 1:4 by volume.

14. The analytical method according to claim 12 or 13, wherein the paraffin melting agent is one or more selected from the group consisting of xylene, limonene, and pinene.

15. The analytical method according to claim 12 or claim 13, wherein the hydrophilizing agent is one or more selected from the group consisting of ethanol, propanol, and acetone.

Images