CN117388035A - Freezing embedding agent and preparation method thereof - Google Patents
Freezing embedding agent and preparation method thereof Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention discloses a freezing embedding agent and a preparation method thereof. The frozen embedding agent consists of the following raw materials in parts by weight: 5.5-16.5 parts of dimethyl sulfoxide, 0.1-1.0 parts of aqueous thickener and 100 parts of water, wherein the aqueous thickener is xanthan gum or guar gum. The frozen embedding agent can improve the quality of frozen sections, and ensure the accuracy of frozen pathological diagnosis in operation and the accuracy of subsequent immunohistochemical detection results. In addition, the preparation method has the characteristics of simple preparation, low cost, simple use and obvious effect.
Description
Technical Field
The embodiment of the invention relates to the technical field of medical tissue sections, in particular to a freezing embedding medium and a preparation method thereof.
Background
Surgical pathology is an important discipline in disease diagnosis and medical research, especially with a decisive role in the diagnosis of neoplastic lesions, called the "gold standard" of diagnosis. However, in many cases there are often cases where a definitive diagnosis cannot be obtained by routine histopathological examination prior to surgery. Thus, pathological diagnosis of lesions during surgery is often required. It is generally known that due to the biophysical characteristics of human tissue, tissue sections cannot be prepared for pathological diagnosis at normal temperature. Therefore, it is generally necessary to take a normalized sample of a tissue sample to be pathologically diagnosed during an operation, and freeze the sample into blocks rapidly to prepare a tissue slice for pathologic diagnosis. Since this method of tableting requires rapid freezing of the tissue mass during the procedure, it is known as "rapid intra-operative frozen pathology", or "intra-operative freezing" for short.
Currently, intra-operative freezing has become a routine pathological diagnostic technique for rapid intra-operative determination of lesion properties, which is crucial for defining lesion properties, assisting the surgeon in determining the surgical mode and the extent of surgical resection. Thus, it is highly appreciated by the surgeon. However, although flash freezing can freeze tissue into pieces and facilitate use of frozen sections with a frozen microtome for pathology diagnosis after staining with hematoxylin-Yin Gong (HE). However, since quick frozen tissue sections are typically around 5 μm thick, they are significantly thicker than conventional paraffin sections. Meanwhile, the tissue brittleness is obviously increased through quick freezing, so that the sections are often discontinuous, wrinkled and cracked. In addition, due to the fact that the moisture content in the human tissue cells is rich, ice crystals with different sizes are inevitably formed in the quick freezing process, and cavitation bubbles and cracks with different sizes are formed on the tissue slices by the formation of the ice crystals, so that the morphological structure of the tissue cells is damaged. Therefore, the quality of the quick frozen section is generally worse than that of a conventional paraffin embedded tissue section, which directly influences or restricts the accuracy of pathological diagnosis of the quick frozen section in operation to a great extent, and is also a main reason of high difficulty and high risk of pathological diagnosis of the quick frozen section in operation.
Furthermore, the rapid freezing causes damage to tissue cell structures caused by formation of ice crystals in tissues, and can also directly damage accurate positioning of molecules in tissue cells, and cause loss and dispersion of corresponding molecules, thereby affecting the accuracy of subsequent detection results such as immunohistochemical staining, and even causing detection failure, missed diagnosis and misdiagnosis. It can be seen that reducing ice crystal formation is critical in improving the quality of intraoperative frozen sections. For this purpose, it is generally suggested to reduce ice crystal formation by sucking the surface water of the tissue with a dry gauze, using a freezing embedding agent, using a frozen microtome with a high freezing rate, or performing quick freezing of the tissue mass with liquid nitrogen. However, the actual effect is not obvious.
At present, the OCT (optimum cutting temperature compound) frozen embedding agent widely used clinically only has the function of providing a soft base for small tissue samples in the rapid frozen section process, increasing the vertical distance between the tissue samples and the surface of a frozen sample holder, reducing the section difficulty, and preventing the blade from directly colliding with the frozen sample holder so as not to cause damage to the blade or the frozen sample holder. Meanwhile, the OCT frozen embedding agent does not have the effect of effectively reducing the formation of ice crystals in tissue cells in the freezing process and reducing the damage of the tissue cells. Thus, the quality of the quick frozen sections is not effectively improved. In addition, the OCT freeze-embedding agent is a water-soluble mixture containing polyethylene glycol and polyvinyl alcohol, wherein the polyvinyl alcohol is an organic compound, and has a chemical formula of [ C 2 H 4 O]n is white flaky, flocculent or powdery solid in appearance. The list of carcinogens published by the world health organization international cancer research institute has listed polyvinyl alcohol as a class 3 carcinogen on day 10 and 27 of 2017. Polyvinyl alcohol has an eye-stimulating effect, and inhalation and ingestion can cause certain harm to the body. Furthermore, although the polyvinyl alcohol in the OCT mixture is soluble in water, the complete dissolution still requires heating to 65-75 ℃, whereas in practice the preparation of flash frozen tissue sections is performed at room temperature (25 ℃). Thus, there must be unequal amounts of polyvinyl alcohol residue in the tissue sample after the OCT is frozen and embedded, which may not only adversely affect subsequent testing, but may also affect the health of the operator by contact.
Therefore, the ice crystal formation in tissue cells of the tissue sample in the rapid freezing process is still difficult to fundamentally reduce by the prior art scheme, so that the quality of the intraoperative frozen section cannot be effectively improved, and the accuracy of intraoperative frozen pathological diagnosis is ensured. Therefore, a new technology for processing tissue samples frozen in operation needs to be explored, and a novel safe and nontoxic frozen embedding agent which can not only effectively reduce the formation of ice crystals in tissue cells in the rapid freezing process, reduce the damage of the tissue cells, increase the continuity of tissue slices in the slicing process, reduce the wrinkles and the cracks of the tissue slices, improve the quality of frozen slices and the accuracy of subsequent immunohistochemical detection, but also provide a soft support base for small tissue samples is developed, and has important clinical value and certain urgency.
Disclosure of Invention
Therefore, the embodiment of the invention provides the frozen embedding agent and the preparation method thereof, and the frozen embedding agent not only can provide a soft base for small tissue frozen sections, but also can effectively reduce the formation of ice crystals in tissue cells in a rapid freezing process, reduce tissue cell damage, increase the continuity of tissues, reduce the occurrence rate of wrinkling and fragmentation of the tissues in the slicing process, improve the quality of frozen sections and the accuracy of subsequent immunohistochemical detection results, and simultaneously has the advantages of simple preparation, low cost, convenient use, no toxicity, no biological safety risk and the like.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
according to a first aspect of an embodiment of the present invention, the present invention provides a frozen embedding agent, which is composed of the following raw materials in parts by weight: 5.5-16.5 parts of dimethyl sulfoxide, 0.1-1.0 parts of aqueous thickener and 100 parts of water, wherein the aqueous thickener is xanthan gum or guar gum.
The invention carries out deep analysis on the formation mechanism of the ice crystals in the tissues and the cells in the rapid freezing process, and considers that the high content of the water in the tissues and the cells is the main reason for forming the ice crystals, so whether the water in the cells of the tissues can be rapidly adsorbed is the key for effectively reducing the formation of the ice crystals in the tissues and the cells.
For this reason, the present invention has been studied on an aqueous thickener having water absorbability. Inorganic thickeners such as bentonite, attapulgite, aluminum silicate and the like are gel minerals which have the characteristic of thixotropic property due to water swelling, but lack adhesiveness, and cannot be sliced after freezing. The polyacrylate and the associative polyurethane thickener are acidic, and the thickening effect can be achieved only by neutralizing the organic thickener with alkali or ammonia water until the pH value is 8-9, so that the organic thickener is not suitable for preparing the intraoperative frozen embedding agent. The cellulose thickener mainly comprises methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and the like, and can be associated with hydrogen bonds of surrounding molecules through a hydrophobic main chain to improve the viscosity of the system, but the cellulose thickener has higher toughness and elasticity and can directly influence the combination between the thickener aqueous solution and frozen tissues. As can be seen, none of the above materials are suitable for preparing frozen embedding agents.
The present invention also finds that natural thickeners, such as plant polysaccharide materials (including pectin, acacia, guar gum, locust bean gum, etc.); algal polysaccharide substances (including agar, alginic acid, carrageenan, etc.); microbial polysaccharides (including xanthan gum, pullulan, etc.); animal substances (including polysaccharide, crust, protein, gelatin, etc.) have good water solubility and thickening effect, and are highly stable to acid, alkali and temperature. Among them, xanthan gum derived from microorganisms and guar gum derived from plants have good water solubility and can exhibit very high viscosity at low mass fraction, so the aqueous thickener of the present application is preferably xanthan gum and guar gum.
Furthermore, the invention discovers that a certain amount of dimethyl sulfoxide is added into the aqueous solution system of the aqueous thickener, and the dimethyl sulfoxide has good tissue cell permeability, is favorable for penetrating into tissue cell gaps and cells and combining with water molecules, thereby reducing the formation of ice crystals inside and outside tissue cells, not affecting the freezing effect of tissue tissues, and having no adverse effect on the subsequent treatment of samples and the detection of molecular targets. Meanwhile, xanthan gum or guar gum belongs to macromolecular polysaccharide substances, and not only can form a good embedding matrix to provide a soft supporting base for a tissue block to be frozen, but also can effectively adsorb moisture on the surface and a section of the tissue block. Therefore, the dimethyl sulfoxide and the xanthan gum or the guar gum are used together, and the dimethyl sulfoxide and the xanthan gum or the guar gum can play a synergistic effect to jointly reduce the formation of ice crystals in tissue cells, so that the quality of frozen slices is effectively improved.
Further, the frozen embedding agent consists of the following raw materials in parts by weight: 11 parts of dimethyl sulfoxide, 1.0 part of an aqueous thickener and 100 parts of water.
According to a second aspect of embodiments of the present invention, there is provided a method of preparing a frozen embedding agent as described above, the method comprising: dimethyl sulfoxide and an aqueous thickener are added into water and mixed evenly.
According to a third aspect of embodiments of the present invention there is provided the use of a frozen embedding agent as described above in intra-operative flash freezing pathology examinations.
According to a fourth aspect of embodiments of the present invention, there is provided a method of preparing a quick frozen tissue section, the method comprising the steps of:
(1) Selecting a frozen sample holder matched with the size of the tissue block;
(2) Dropwise adding the frozen embedding agent on the frozen sample holder;
(3) Placing the tissue block on the frozen embedding agent, naturally settling the tissue block into the frozen embedding agent, and then dripping a small amount of frozen embedding agent on the surface of the tissue block until the tissue block is completely covered;
(4) And placing the frozen sample support with the tissue blocks wrapped by the frozen embedding agent on a freezing table for quick freezing, and then slicing to prepare the quick frozen tissue slices.
Further, in the step (4), the quick freezing temperature is-45 ℃ to-16 ℃ and the time is 5-10 minutes. The rapid freezing temperature is set according to the tissue type, and the rapid freezing temperature of soft tissues is typically-16 ℃ to-20 ℃, and the rapid freezing temperature of tissues with high fat content can be set to-30 ℃ to-45 ℃.
The embodiment of the invention has the following advantages:
the frozen embedding agent provided by the invention utilizes the characteristic that an aqueous thickener (preferably xanthan gum or guar gum) aqueous solution is in a gel state at normal temperature and can be quickly coagulated into blocks at low temperature, so that not only can good support be provided for small tissue blocks, the collision between a blade and equipment in the slicing process be reduced, but also the frozen embedding agent can quickly infiltrate into a tissue gap and be frozen into a whole with the tissue blocks, thereby ensuring the continuity and the integrity of quick frozen tissue slices and reducing the occurrence rate of wrinkling and fragmentation of the tissue slices. Meanwhile, the water absorption performance of dimethyl sulfoxide and an aqueous thickener (preferably xanthan gum or guar gum) and the permeability of the dimethyl sulfoxide are utilized to rapidly adsorb water inside and outside tissue cells, so that the formation of ice crystals in tissue cells of a tissue block in a rapid freezing process is effectively reduced, the mechanical damage of the ice crystals to the tissue cells and the loss of normal subcellular localization, dispersion and even loss of intracellular biomolecules caused by the mechanical damage of the ice crystals are reduced, and the quality of rapid frozen sections in an operation is improved.
The components such as dimethyl sulfoxide, xanthan gum or guar gum used by the frozen embedding agent are harmless, nontoxic and easily soluble in water, are stable to temperature, acid, alkali, salt, oxidant and protease, do not influence the structure, activity and functional state of biological molecules in tissue cells, do not influence subsequent tissue treatment and dyeing, and do not pollute the environment, and influence the health of operators.
The frozen embedding agent has the advantages of simple preparation, low cost and convenient use.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
FIG. 1 is a schematic illustration of a method of using a frozen embedding medium of the present invention, 1-frozen sample holder; 2-dropwise adding a freezing embedding agent; 3-organizing the blocks; 4-supplementing the dropwise added frozen embedding agent.
FIG. 2 is a photomicrograph of a quick frozen section (HE staining, x 200) provided by the present invention, A: OCT frozen embedding agent; b: the frozen embedding agent of example 1 was used; c: the frozen embedding agent of example 2 was used; d: the frozen embedding agent of example 3 was used.
FIG. 3 is a micrograph of immunohistochemical staining of paraffin-embedded tissue after flash freezing (immunohistochemical staining VENTANA method, ×200), A, B, C is the expression of ER, PR and HER2, respectively, in conventional paraffin-embedded tissue; D. e, F expression of ER, PR and HER2, respectively, in paraffin-embedded tissue after flash freezing treated with OCT embedding agent; G. h, L are the expression of ER, PR and HER2, respectively, in paraffin-embedded tissue following flash freezing with the freezing embedding treatment of the freezing embedding medium of example 2.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a freezing embedding agent, which is prepared by the following steps:
5.5g dimethyl sulfoxide and 1.0g xanthan gum were added to 100mL water and mixed well.
Example 2
The embodiment provides a freezing embedding agent, which is prepared by the following steps:
11.0g of dimethyl sulfoxide and 1.0g of xanthan gum were added to 100mL of water and mixed well.
Example 3
The embodiment provides a freezing embedding agent, which is prepared by the following steps:
16.5g dimethyl sulfoxide and 1.0g xanthan gum were added to 100mL water and mixed well.
Example 4
The embodiment provides a freezing embedding agent, which is prepared by the following steps:
11.0g of dimethyl sulfoxide and 1.0g of guar gum were added to 100mL of water and mixed well.
Test example 1
To verify the practical application effect of the frozen embedding agent, 131 samples of rapid frozen pathological examination in the simulated operation are collected, wherein 68 samples of breast cancer are collected. The frozen embedding agent and the traditional OCT frozen embedding agent are adopted to embed the tissue blocks, the using effect of the frozen embedding agent is compared, meanwhile, the subcellular localization and expression of ER, PR and HER2 in the quick frozen tissue blocks of 68 breast cancer specimens are detected by adopting immunohistochemical staining, and the influence of the frozen embedding agent on the immunohistochemical staining result of the quick frozen tissues is analyzed.
1 main instrument
Frozen microtomes (LEICA CM 1950), tissue dehydrators (DAKEWE HP 300), full-automatic immunohistochemistry apparatus (rochantenchmark), paraffin microtomes (LEICA CV 5030), full-automatic HE stainers (DAKEWE DP 260), and wafer sealers (DAKEWE).
2 main reagents
OCT cryo-embedding agents were purchased from Leica (Leica), anti-ER antibody (C6H 7), anti-PR (antibody C12A 28), anti-HER 2 antibody (EP 3) from Sainort, and VENTANA secondary antibody systems from Roche.
3 method
3.1 Effect of frozen embedding agent on quick frozen tissue sections
1) Sample material
The rapid freezing pathological examination specimens in 131 cases of operation for inspection are subjected to standard material drawing, 4 tissue blocks with approximately similar sizes, thicknesses and thicknesses are respectively cut on the premise of ensuring that pathological diagnosis is not affected, and the length and the width of the tissue blocks are smaller than 1.5cm, and the thickness is smaller than 0.3cm. Then, after the rest tissue specimens are put into 10% neutral buffer formalin solution for fixation, paraffin wax blocks are prepared by standard material drawing, dehydration and paraffin embedding.
2) Quick freezing and sectioning of tissue pieces
And respectively dripping OCT frozen embedding agent and 1-2ml of frozen embedding agent of examples 1-3 on 4 spare frozen sample holders, respectively placing the cut tissue blocks on the frozen sample holders, and respectively dripping a small amount of corresponding frozen embedding agent on the surfaces of the tissue blocks after the tissue blocks naturally subside until the tissue blocks are completely covered. Then, the frozen sample holder together with the tissue blocks embedded with the frozen embedding agent are placed on a freezing table in a frozen microtome for quick freezing, and then conventional quick frozen microtome and HE staining are performed by using the frozen microtome. The tissue blocks after the quick frozen sections are fixed by 10% neutral formalin, dehydrated and paraffin embedded, and the corresponding paraffin embedded paraffin blocks after the quick frozen sections are prepared for standby.
3) Observation under microscope
The quality of frozen sections treated with different frozen embedding agents, including staining of the sections, tissue cell morphology and structural integrity, continuity, fold and fragmentation rate, number of cavitation bubbles, etc., were observed under a microscope. The number of cavitation bubbles in 10 high power fields (400X) per slice was counted and the average (in/HFP) was recorded. Results were analyzed using the Wilcoxon signed rank test.
3.2 Effect of frozen embedding agent on tissue immunohistochemical staining results after quick freezing
1) Wax block selection
68 breast cancer specimens were selected from 131 flash frozen specimens in the simulated surgery of the stock, from which the flash frozen paraffin-embedded tissue blocks embedded with the frozen embedding agent of example 2 were selected, and the flash frozen paraffin-embedded tissue blocks embedded with the OCT frozen embedding agent and the normal paraffin-embedded tissue blocks without flash freezing were used as controls.
2) Immunohistochemical staining and interpretation of results
Immunohistochemical staining was performed using a rogowski VENTANABench Mark fully automatic immunohistochemical machine, by observing the subcellular localization and expression of ER, PR, HER2 in mammary tissue cells under a microscope (wherein ER, PR immunohistochemical staining results are expressed in terms of positive percentages, HER2 immunohistochemical staining results are interpreted according to WHO interpretation guidelines version 2018, expressed as 0, 1+, 2+, 3+, and the effect of different frozen embedding agents on the rapid frozen tissue immunohistochemical staining results was analyzed and evaluated using Wilcoxon symbol rank test.
Results 4 results
4.1 Effect of frozen embedding agent on quick frozen sections
The results show that the frozen tissues embedded by the frozen embedding agent provided by the embodiment of the invention have no difference with the tissues embedded by the existing OCT frozen embedding agent in the aspects of freezing time and slicing difficulty.
Morphological observations were made under a microscope on frozen sections treated with different frozen embedding agents. The results showed that the OCT frozen embedded frozen sections stained well but the tissue structure was incomplete on some sections, the wrinkles and interstitial fissures were evident in the sections, and unequal amounts of clear vacuoles (remnants left after ice crystal dissolution) in the nuclei were visible in localized areas of the sections, averaging 18.3 per high power field of view per HFP (fig. 2A). Whereas the quick frozen sections of the tissue blocks respectively embedded with the frozen embedding agents of examples 1-3 were well stained, the tissue structure on the sections was substantially continuous and complete, the occurrence of wrinkles, interstitial fissures and breakage in the sections was significantly reduced, and the average number of interstitial and nuclear vacuoles was 7.2/HFP, 3.5/HFP and 4.6/HFP, respectively (B, C, D in FIG. 2). The Wilcoxon signed rank test shows that the quality of the quick frozen tissue slice of the frozen embedding agent embedded tissue block is obviously superior to that of the quick frozen tissue slice of the OCT frozen embedding agent embedded tissue block, and the difference has statistical significance (P is less than 0.001).
4.2 Effect of frozen embedding agent on the immunohistochemical results of rapidly frozen tissue
Referring to fig. 3, a and B, C are the expression of ER, PR and HER2 in conventional paraffin-embedded tissues, respectively, and the tissue cell structure is clear under a microscope, wherein ER (a) and PR (B) are expressed by strong positive nuclei, and HER2 (C) is expressed by strong positive membranes; D. e, F is the expression of ER, PR and HER2 in paraffin embedded tissues after quick freezing by adopting OCT freezing embedding agent, the tissue cell structure is fuzzy under a microscope, ER (D) and PR (E) are expressed by weak positive cell nuclei, the positive rate is obviously reduced, HER2 (F) is expressed by weak positive incomplete cell membranes, and cytoplasmic staining occurs; G. h, L are the expression of ER, PR and HER2 respectively in paraffin-embedded tissues after quick freezing by embedding treatment of the freezing embedding agent of the example 2, the structure of tissue cells is basically clear under a microscope, ER (G) and PR (H) are expressed by strong positive nuclei, and HER2 (L) is expressed by strong positive and basically complete cell membranes.
The results show that the tissue structure in the paraffin-embedded tissue immunohistochemical stained sections was continuous, free of obvious interstitial and intracellular cavities, and also free of obvious non-specific background staining in 68 cases of the paraffin-embedded tissue after flash freezing by the frozen embedding agent of example 2, as compared to the paraffin sections of the paraffin-embedded tissue after flash freezing by OCT frozen embedding agent treatment. ER and PR show clear nuclear localization in ER and PR positive expression cases, HER2 shows clear cell membrane coloring in HER2 positive expression cases, and no obvious intracytoplasmic diffusion exists. Meanwhile, the positive staining intensity of ER, PR, HER is obviously better than that of paraffin sections treated by OCT frozen embedding agent in corresponding cases after quick freezing. The differences were all statistically significant (P < 0.001) as demonstrated by Wilcoxon signed rank test.
ER, PR, HER2 shows a different degree of attenuation in the expression intensity in paraffin-embedded immunohistochemical stained sections after 68 quick freezing with the frozen embedding agent of example 2 compared to the corresponding conventional paraffin section immunohistochemical staining results, but has substantially no effect on the interpretation of ER, PR, HER2 immunohistochemical staining results (p=0.083 > 0.05).
Therefore, the frozen embedding agent provided by the invention not only provides good support for frozen small tissues, but also can make up for the defect that the existing OCT frozen embedding agent can not reduce the formation of ice crystals in tissue cells in the rapid freezing process to a great extent, and the water absorption performance of dimethyl sulfoxide and an aqueous thickener (preferably xanthan gum or guar gum) and the high permeability of dimethyl sulfoxide adsorb the water inside and outside tissue cells, so that the formation of ice crystals in tissue cells in the freezing process in operation and the damage of the ice crystals to subcellular structures in tissue cells in the freezing process in the operation are effectively reduced, and the conditions of dispersion and even deletion of intracellular molecules caused by the formation of normal subcellular localization are also obviously improved, the continuity and the integrity of tissues in the traditional rapid frozen slicing process are reduced, the occurrence rate of tissue slice wrinkles and fragmentation are reduced, the frozen slice quality is obviously improved, and the accuracy of frozen pathological diagnosis in operation and the accuracy of a subsequent immunohistochemical detection result are ensured.
The dimethyl sulfoxide and the aqueous thickener adopted by the frozen embedding agent provided by the invention, such as xanthan gum, guar gum and the like, have no toxic or side effect, are easy to dissolve in water at normal temperature, and can not cause any adverse effect on molecular target detection results such as subsequent tissue treatment, immunohistochemical staining and the like, and the physical health of operators can not be endangered. In addition, the frozen embedding agent provided by the invention is convenient to prepare, low in cost and simple and convenient to use and operate.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (6)
1. The freezing embedding agent is characterized by comprising the following raw materials in parts by weight: 5.5-16.5 parts of dimethyl sulfoxide, 0.1-1.0 parts of aqueous thickener and 100 parts of water, wherein the aqueous thickener is xanthan gum or guar gum.
2. The frozen embedding medium according to claim 1, characterized in that the frozen embedding medium consists of the following raw materials in parts by weight: 11 parts of dimethyl sulfoxide, 1.0 part of an aqueous thickener and 100 parts of water.
3. A method of preparing a frozen embedding medium as claimed in claim 1 or 2, characterized in that the method comprises: dimethyl sulfoxide and an aqueous thickener are added into water and mixed evenly.
4. Use of a frozen embedding agent according to claim 1 or 2 for intra-operative rapid frozen pathology examination.
5. A method of preparing a quick frozen tissue section, the method comprising the steps of:
(1) Selecting a frozen sample holder matched with the size of the tissue block;
(2) Dropping the frozen embedding agent according to claim 1 or 2 onto the frozen sample holder;
(3) Placing the tissue block on the frozen embedding agent, naturally settling the tissue block into the frozen embedding agent, and then dripping a small amount of frozen embedding agent on the surface of the tissue block until the tissue block is completely covered;
(4) And placing the frozen sample support with the tissue blocks wrapped by the frozen embedding agent on a freezing table for quick freezing, and then slicing to prepare the quick frozen tissue slices.
6. The method of preparing a quick frozen tissue slice according to claim 5, wherein in step (4), the quick freezing temperature is-45 ℃ to-16 ℃ for 5-10 minutes.
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