KR100578034B1 - Stain and staining method for staining of frozen section - Google Patents

Abstract

The present invention relates to a dye composition for freezing section staining and freezing section dyeing method using the same. The present invention, 2 to 6 mg of urea; Distilled water of 100 to 150 kPa; 150 to 300 kPa of isopropanol; And 0.6 to 1.0 mg of toluidine blue; and is characterized by consisting of Eosin Y having a concentration of 0.3 to 0.8% in 100% isopropanol solvent. In addition, it is characterized by dyeing the frozen section through a unique staining method using the same. According to the present invention, the accuracy of optical observation on the dyed frozen section tissue, and thus the more precise selection of the target area and the improved material retention inside the tissue section, make it more suitable for research work involving optical observation and microdissection apparatus. It offers the advantage of obtaining efficient research results.

Freeze Section, Toluidine Blue, Eosin, Dyeing, Optical Microscope

Description

Dye composition for freezing section staining and freezing section staining method using the same {Stain and staining method for staining of frozen section}

1 to 4 are photographs observed through an optical microscope after the dyeing process using a dye composition comprising toluidine blue and eosin Y according to the present invention.

5 is a photograph showing the results of measuring RNA quality in tissue after dyeing frozen sections using the dye composition of toluidine blue and eosin Y according to the present invention.

The present invention relates to a dye composition for cryosection staining and a cryosection staining method using the same, and more particularly, toluidine blue for cryosection staining for improving the preservation of macromolecules contained in cryosection tissue. It relates to a dye composition comprising and a freeze section dyeing method using the same.

In the field of optical microscopy or microdissection genomics, the morphological preservation of the biological tissues employed as the target specimens is well preserved, so that the microscopic observation is not only easy, but also the various giants contained in the biological tissues. Preservation of molecular material such as DNA, RNA, protein, etc. should be made. If the shape and the material itself are not properly preserved within these target specimens, it will be difficult to make an accurate diagnosis when examining the optical microscope, or it may be difficult to discriminate micro lesions. If the molecular material is destroyed, there may be a problem that the reliability of the results is not sufficiently secured.

Conventionally, in order to observe a specific tissue section of a biological tissue by an optical microscope, a slide for observation in which a tissue specimen is mounted has been manufactured through several steps in advance. Step by step in the fabrication process of the tissue specimens seated on such slides, after extracting a portion of the necessary tissue from a biological source, such as animals, plants and microorganisms, including humans, fixing the tissue section (fixing), tissue section Dehydration step to remove water remaining in the inside, Clearing step to replace the alcohol penetrated into the tissue section in the dehydration process with xylene, Clearing the paraffin solution to include the paraffin solution in the tissue form Each is subjected to an embedding step of manufacturing. Subsequently, a section of the paraffin block is sliced to a predetermined thickness, and the tissue sections sliced into a flake state are mounted on the upper slide, and then, a predetermined dye is used for intracellular components for optical microscope observation. It is progressing to the staining step (staining) to perform the staining.

However, the conventional process for preparing tissue sections has good morphological preservation, but the macromolecules inside the tissues during the preparation of the tissue sections, during the fixation or paraffin embedding process. It has the disadvantage of being damaged.

By the way, when the frozen section is prepared using a cryo-tissue sectioner (crymicrotome), there is an advantage that the macromolecular substances in the tissue are well preserved because there is no need for the fixation or paraffin embedding process. However, as the freezing process progresses, ice crystals are formed inside the microscopic tissue sections, and thus the cryosection method is limited to maintain the morphological preservation. Despite these shortcomings, the field of genomics studies on specimens subjected to microscopic ablation has had a morphologically relatively small effect because the preservation of macromolecules significantly affects the analytical results, resulting in a smaller size of ice crystals. Inevitably select and use a rapid freezing method that can drive the mad. In addition, it is recognized that the preservation of macromolecules in tissues is improved, but various endogenous enzymes capable of destroying macromolecules are also preserved intact. Special care has to be taken because it can be activated and destroy macromolecules. In particular, in order to observe the frozen section through an optical microscope, it is essential to stain the frozen section. Most of the dyeing materials currently used are water-soluble dyes such as hematoxylin and eosin. I'm using. Therefore, there is an inevitable process of exposing the frozen sections to water during the dyeing process, and endogeneous macromolecular destructive enzymes such as DNase, RNase, and protease. Protease) may cause a problem that macromolecules, such as DNA, RNA, proteins, and the like in the frozen fragments are destroyed during staining. For example, differences may occur depending on the type of tissue, but in the case of rat intestinal tissues, it is known that more than 90% of RNA is destroyed after exposure to water for 1 minute. As a method for reducing the dyeing time is used. However, the shortening of the dyeing time is a problem that it is difficult to observe the exact shape when the optical microscope observation as well as the degree of dyeing is not made effectively because the dyeing.

In addition, conventional staining method using hematoxylin and Eosin (Hemtoxylin & Eosin) is the most commonly used staining method for tissue sections (including freeze section) staining, hematoxylin combines with DNA and RNA to make the nucleus appear blue, Eosin binds to the proteins that make up the cytoplasm and is stained red. In particular, hematoxylin stains in the nucleus appear blue, rather than binding directly to the nucleus in the form of hematoxylin, but the oxidized form of hematein (haematein) is a mordant with aluminum salts or iron salts. It is known to bind to nuclear chromatin together as a complex. Therefore, it has been pointed out that the staining method using hematoxylin may adversely affect the material retention and quality of DNA and RNA.

Therefore, no matter how well preserved macromolecules are stored in the frozen sections, they must be dyed to observe them morphologically. This problem can be solved because the macromolecules can be destroyed by the staining process or the dye solution. There is a need to be solved, and research to find a solution to this has been continuously conducted in the related field, and the present invention has been devised under such a technical background.

Based on the above-mentioned problems of the prior art, the present invention provides a method for improving the optical resolution of a tissue section at the time of microscopic observation. It should be able to minimize the destruction of macromolecules by inhibiting activation. 2) Extraction process of macromolecules such as DNA, RNA and protein, which are well preserved in the excised tissue sections collected by microscopic observation and microscopic ablation process. In order to achieve a number of technical problems such as not to have any effect on the same, in order to achieve the technical problem, it is an object of the present invention to provide a dye composition and a freeze section dye method for dyeing the frozen sections.

Dye compositions for cryosection staining to achieve the object of the present invention, 2 to 6 mg of urea; Distilled water of 100 to 150 kPa; 150 to 300 kPa of isopropanol; And 0.6 to 1.0 mg of toluidine blue.

On the other hand, cryosection staining method for achieving the object of the present invention, (a) fixing the frozen piece specimen seated on the slide using ethanol; (b) immersing the frozen section specimen fixed with ethanol in isopropanol; (c) depositing the frozen piece specimen deposited in the isopropanol to a predetermined toluidine blue; (d) first washing the frozen piece specimen deposited in the toluidine blue solution with ethanol; (e) immersing the frozen section of the first wash with ethanol in a predetermined eosinwai; (f) second washing of the frozen piece specimen deposited on the eosin Y with ethanol; (g) immersing the frozen section of the second wash with ethanol in a mixed solution of isopropanol and xylene; (h) depositing the frozen piece specimen deposited in the mixed solution of isopropanol and xylene in xylene; And (i) drying the frozen piece specimen deposited in xylene at room temperature.

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At this time, each step of the frozen section staining method is preferably carried out as follows. That is, in the step (a), the frozen section specimens seated on the slide are first fixed at 0.5 to 2 minutes using 100% ethanol at -15 ° C to minus 25 ° C, and then 0.5 to 2 minutes at room temperature at 100% ethanol. It is preferable to proceed to the second fixing step, the step (b) is preferably carried out in the step of immersing the frozen specimen fixed in ethanol 5 to 15 times in 70% isopropanol in the step (a), Step (c) is preferably proceeded to the step of immersing the frozen specimen deposited in isopropanol in step (b) in 0.1 to 0.3% toluidine blue solution for 15 to 30 seconds, the step (d) is (c) Preferably, the step of washing the frozen specimen deposited in toluidine blue solution in the step of first washing with 100% ethanol is preferred, and the step (e) is performed in the step (d) of the frozen washing specimen first washed with ethanol. 0.3 to 0.8% eosin Y is preferable to proceed to the step of immersion from 15 to 30 seconds (100% isopropanol solvent). In addition, the step (f) is preferably carried out in the step of washing the frozen specimen specimen deposited with Eosin Y in step (e) three to eight times with 100% ethanol, step (g) is the ( It is preferable to proceed with the step of immersing the frozen specimens washed with ethanol in step f) for 15 to 25 seconds in a mixed solution of isopropanol and xylene 1:18 to 1:22, the step (h) In the step (g), it is preferable to proceed with the step of immersing the frozen sample specimen deposited in the mixed solution of isopropanol and xylene in xylene 1 to 3 times for 15 to 25 seconds each time. Finally, the step (i) is preferably carried out to air-dried for 4 to 6 minutes the frozen piece specimen deposited in xylene in the step (h).

Numerical limitation of each of the dye compositions for freezing section dyeing according to the present invention and the dyeing method for the frozen section using the same represents the technical and economic limitations that can achieve the optimal effect in the practice according to the present invention. Therefore, when exceeding the range of these numerical limitations, it is obvious that it is difficult to expect the effect predicted by the present invention.

Hereinafter, the preparation of the dye composition for freezing section dyeing according to the present invention will be described in detail with reference to Examples to explain the dyeing method for the frozen section, in detail with reference to the accompanying drawings to help the understanding of the invention. Let's do it. However, embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

First, the process of preparing a dye composition comprising toluidine blue for use for nuclear staining will be described. Conventional hematoxylin stains may affect the quality of DNA or RNA, as described above, while toluidine blue stains are relatively weak as well as reversible and thus reversible. It is known to have less effect. In addition, toluidine blue is generally water-soluble and has been widely used for mono-dye by dissolving in water. However, since toluidine blue is soluble in isopropanol, the dye can be thickened by adding urea, particularly by changing the solvent to isopropanol.

The dye composition for dyeing the frozen sections described below is 0.2% toluidine blue dye solution, which contains 4 mg of urea, 120 kV distilled water, 280 kV isopropanol, 0.8 mg of toluidine blue (Sigma T3260, dye contents). 80%). First, the prepared urea and toluidine blue are dissolved in water, isopropanol is added to 70%, and then filtered and stored for a long time.

Next, the process of preparing a dye composition comprising Eosin Y for use for cytoplasmic staining is as follows. That is, 0.5% Eosin Y (Sigma: Eosin Y disodium salt E-6003) was dissolved in 100% isopropanol solvent.

Finally, the method of dyeing frozen sections using the prepared dye composition for nucleation and dye composition for cytoplasmic dyeing will be described sequentially. Please explain)

① The frozen section specimens seated on the slides are immediately fixed in 100% ethanol at -20 ° C for 1 minute, and then fixed in 100% ethanol at room temperature for 1 minute. At this time, water or moisture inherent in the tissue specimen is removed at the same time as the fixing. This process is not only good for increasing the morphological preservation of frozen section specimens by moving them from room temperature to -20 ° C stepwise, but also for the inactivation of enzymes that can break through the macromolecular material of the frozen section specimens with alcohol. It is an important process. ② dip 10 times in 70% isopropanol. This process is necessary to accommodate frozen section specimens because the next staining process is dissolved in 70% isopropanol. ③ Soak in 0.2% toluidine blue solution for 15 to 30 seconds. ④ Wash with 100% ethanol. ⑤ Soak 0.5% Eosin Y dissolved in 100% isopropanol for 15-30 seconds. ⑥ Wash 5 times with 100% ethanol. (7) Soak for 20 seconds in a solution of isopropanol / xylene = 1: 20. ⑧ Soak in xylene twice for 20 seconds each. ⑨ Dry at room temperature for 5 minutes.

The treatment of xylene in step ⑧ is a treatment process to prevent this because the decolorization phenomenon is caused by the water action by isopropanol immediately after passing through 100% isopropanol in the case of the frozen section, air drying immediately.

After the dyeing process using the dye composition for freezing section dye prepared as described above, through the optical microscope observation of the dyed frozen section, it was confirmed that the morphological material preservation, the result is shown in Figures 1 to 4 Indicated.

1 to 4 are photographs observed through an optical microscope after the dyeing process using a dye composition comprising toluidine blue and eosin Y according to the present invention.

1 is a photograph showing the appearance after staining the frozen section tissue of the human mucosa. In particular, Figure 2 is an enlarged picture of Figure 1, it can be seen that all the cells are observed very clearly chromatin pattern of the nucleus.

FIG. 3 is a photograph observed using an optical microscope after gastric cancer cells infiltrating lymph nodes are stained by the dye composition according to the present invention. In particular, FIG. 4 is an enlarged photograph of FIG. It can be confirmed that it is observed.

5 is a chromatographic photograph showing the results of measuring RNA quality in tissue after dyeing frozen sections using a dye composition of toluidine blue and eosin Y according to the present invention. For this purpose, RNA quality was measured using an RNA 6000 nano analyzer (manufactured by Agilent Technologies). From the photograph of FIG. 5, both the upper band 28S band 51 and the lower band 18S band 52 are observed to remain vividly consistent with the initial stage despite the passage of time, and thus, the dye composition and dyeing according to the present invention. It is apparent that the method does not affect macromolecules including RNA in tissues, so that it is possible to more reliably understand that the material conservation and the shape conservation are good.

Optimal embodiments of the present invention described above have been disclosed. Although specific terms have been used herein, they are used only for the purpose of describing the present invention in detail to those skilled in the art and are not intended to limit the scope of the present invention as defined in the claims or the claims.

According to the dye composition for the frozen section according to the present invention, the frozen section dyeing method using the same, the following specific advantages can be confirmed.

First, by ensuring sufficient optical resolution of tissue sections during microscopic observation, precise selection for microscopic observation of the tissue sections and laser ablation of the microscopic sections was possible.

Second, the macromolecules contained in the tissue sections during staining process can be minimized by damages caused by exposure to air or moisture, thereby improving the preservation of various macromolecules in the tissue sections, thereby ensuring the reliability of subsequent studies. It became. In particular, since the dye composition used for cryosection staining is a non-aqueous alcoholic solution, activation of macromolecules, particularly endogenous RNases, which may be present in tissue sections, may be exposed to moisture. It is preferable because it can prevent.

Third, DNA, RAN, which is a macromolecular material well preserved in the resected tissue sections collected by microscopic observation as well as not degeneration of the macromolecules (macromolecule) of the tissue sections after staining process. And since it has no influence on the extraction process of substances, such as a protein, it is preferable that the possibility of commercialization is high.

Ultimately, the accuracy of the observations, resulting in more precise selection of target areas and improved material preservation inside tissue sections, provide the advantage of more efficient research results in research work involving optical observation and microdissection devices. .

Claims (12)

2-6 mg of urea; Distilled water of 100 to 150 kPa; 150 to 300 kPa of isopropanol; And 0.6 to 1.0 mg of toluidine blue; dye composition for freeze section dye, characterized in that it comprises a composition. delete In the method of dyeing frozen sections, (a) fixing the frozen section specimens seated on the slide with ethanol; (b) immersing the frozen section specimen fixed with ethanol in isopropanol; (c) depositing the frozen piece specimen deposited in the isopropanol to a predetermined toluidine blue; (d) first washing the frozen piece specimen deposited in the toluidine blue solution with ethanol; (e) immersing the frozen section of the first wash with ethanol in Eosin Y; (f) second washing of the frozen piece specimen deposited on the eosin Y with ethanol; (g) immersing the frozen section of the second wash with ethanol in a mixed solution of isopropanol and xylene; (h) depositing the frozen piece specimen deposited in the mixed solution of isopropanol and xylene in xylene; And (i) air drying the frozen piece specimen deposited on the xylene. According to claim 3, wherein the step (a) is frozen frozen specimens seated on the slide immediately after the first fixed for 0.5 to 2 minutes using 100% ethanol at minus 15 to minus 25 ℃, then in 100% ethanol at room temperature Freeze section staining method characterized in that the step of secondary fixation for 0.5 to 2 minutes. The method of claim 3, wherein step (b) comprises the step of immersing the frozen specimen fixed in ethanol 5 to 15 times in 70% isopropanol in step (a). According to claim 3, wherein the step (c) is a frozen section characterized in that the step of immersing the frozen specimen deposited in isopropanol in step (b) in 0.1 to 0.3% toluidine blue solution for 15 to 30 seconds Dyeing method. The method of claim 3, wherein step (d) comprises performing the first washing of the frozen piece specimen deposited in the toluidine blue solution in step (c) with 100% ethanol. According to claim 3, wherein step (e) proceeds to the step of immersing the frozen specimen first washed with ethanol in step (d) in 0.3 to 0.8% Eosin Y (solvent 100% isopropanol) for 15 to 30 seconds. Freeze section staining method characterized in that. According to claim 3, wherein the step (f) is frozen section staining characterized in that the step of washing the frozen specimens deposited with Eosin Y in step (e) three to eight times with 100% ethanol 2 times. Way. The method of claim 3, wherein step (g) is performed for 15 to 25 seconds in the mixed solution in which the mixing ratio of isopropanol and xylene is 1:18 to 1:22 in the frozen section specimen washed with ethanol in step (f). Cryosection staining method, characterized in that to proceed to the deposition step. According to claim 3, wherein the step (h) proceeds to the step of immersing the frozen specimen specimen deposited in the mixed solution of isopropanol and xylene in xylene 1 to 3 times for 15 to 25 seconds each time in step (g) Freeze section staining method characterized in that. The method of claim 3, wherein step (i) comprises the step of drying the frozen piece specimen deposited in xylene in step (h) at room temperature for 4 to 6 minutes.

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