CN114097766A - Specimen embedding liquid and preparation method thereof - Google Patents
Specimen embedding liquid and preparation method thereof Download PDFInfo
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- CN114097766A CN114097766A CN202111470891.3A CN202111470891A CN114097766A CN 114097766 A CN114097766 A CN 114097766A CN 202111470891 A CN202111470891 A CN 202111470891A CN 114097766 A CN114097766 A CN 114097766A
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- gelatin
- solution
- active agent
- specimen
- embedding
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- 239000007788 liquid Substances 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims description 7
- 108010010803 Gelatin Proteins 0.000 claims abstract description 46
- 239000008273 gelatin Substances 0.000 claims abstract description 46
- 229920000159 gelatin Polymers 0.000 claims abstract description 46
- 235000019322 gelatine Nutrition 0.000 claims abstract description 46
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 46
- 239000013543 active substance Substances 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000000049 pigment Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 23
- 239000012530 fluid Substances 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 7
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 4
- 229940043267 rhodamine b Drugs 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 2
- 235000015110 jellies Nutrition 0.000 claims 1
- 239000008274 jelly Substances 0.000 claims 1
- 238000007710 freezing Methods 0.000 abstract description 21
- 230000008014 freezing Effects 0.000 abstract description 21
- 239000000725 suspension Substances 0.000 abstract description 6
- 239000000084 colloidal system Substances 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000005057 refrigeration Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention relates to a specimen embedding liquid and a device, wherein the specimen embedding liquid comprises 3-5% of gelatin, 1-3% of an active agent, no more than 1.5 parts per million of pigment, and the balance of water. Adding gelatin, active agent and pigment at a given ratio into water as base solution to form embedding solution which is colloid at 10 deg.C and has reduced brittleness after freezing; the active agent is used for forming the added components in the embedding liquid into a suspension state, so that the brittleness is further reduced after freezing, and ice blocks cannot be layered; the pigment is used for distinguishing the boundary between the specimen in the embedding liquid and the ice block around the specimen.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a specimen embedding liquid and a preparation method thereof.
Background
The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.
The frozen section is a method for rapidly cooling tissues to a certain hardness under a low temperature condition and then slicing, the whole sample is frozen and then sliced, and the image data of each slice is obtained and is reconstructed by 3D to form a virtual digital image which is used in the fields of medical teaching, research and the like, so that the sample resources can be saved, and the method is gradually popularized in the fields of medical research and teaching.
The specimen is an irregular object, is difficult to position on slicing equipment when directly placed, needs to be embedded, and is wrapped by embedding liquid, so that the specimen forms a regular object after being frozen, and the positioning in the slicing process is facilitated.
The main component of the existing embedding liquid is water, and the frozen ice blocks are hard and brittle and are easy to crack in the processes of carrying and slicing the specimens, so that the regular shapes of the ice blocks are lost; in the freezing process of embedding liquid, the composition of current embedding liquid is difficult for keeping the suspended state, has the settlement phenomenon in freezing process, causes the ice-cube fragility inconsistent after freezing, makes the section of cutting present turbid layering phenomenon under the supernatant simultaneously, can lead to follow-up section image recognition, acquisition and the reduction of 3D reconstruction's precision because of section image quality is not good.
Meanwhile, the color difference between the embedding liquid with water as a main component and the color of the boundary of the specimen is small, the embedding liquid is blurred with the boundary line of the specimen after being frozen, and the boundary of the specimen is difficult to distinguish in the subsequent 3D reconstruction process of the slice image, so that the width and height data of the section of the specimen are difficult to obtain.
Disclosure of Invention
In order to solve the technical problems in the background technology, the invention provides a specimen embedding liquid and a preparation method thereof, wherein water is used as a base liquid, gelatin, an active agent and pigment with set proportion are added, the formed embedding liquid is in a colloid state at 10 ℃ by utilizing the gelatin, and the brittleness is reduced after freezing; the active agent is utilized to enable the components in the embedding liquid to be in a suspension state, so that the brittleness is further reduced after freezing, and ice blocks cannot be layered; the pigment is used for distinguishing the boundary between the specimen in the embedding liquid and the ice block around the specimen.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a specimen embedding liquid, which comprises 3-5% of gelatin, 1-3% of active agent, no more than 0.15% of pigment and the balance of water.
The active agent is sodium dodecyl sulfate.
The pigment comprises methylene blue or rhodamine B.
The embedding liquid is jelly-shaped when cooled to 10 ℃.
The second aspect of the present invention provides a method for preparing the above embedding liquid, comprising the steps of:
gelatin and water were mixed in a ratio of 1: 5, preparing a gelatin solution by soaking, heating and stirring the gelatin solution;
according to the following steps: 500 of active agent aqueous solution;
dropwise adding the gelatin solution into the active agent aqueous solution, stirring to obtain a mixed solution, and heating the mixed solution to form a fluid state;
adding pigment into the fluid mixed solution, and cooling to normal temperature to form the prepared embedding solution.
The gelatin is soaked in water for at least 1 hour.
The gelatin is soaked in water at room temperature, and the aqueous solution of the active agent is prepared at room temperature.
After the gelatin solution was heated and stirred to a gel state, it was added dropwise to the aqueous active agent solution.
The gelatin solution is heated in a water bath mode, and the mixed solution is heated in a water bath mode.
The heating temperature of the gelatin solution is not lower than 45 ℃, and the heating temperature of the mixed solution is not lower than 45 ℃.
Compared with the prior art, the above one or more technical schemes have the following beneficial effects:
1. the addition of the gelatin can improve the viscosity of water, reduce the brittleness of ice cubes and ensure the stable later processing.
2. When gelatin concentration is less than 3%, the whole fragility of ice-cube is too big, the phenomenon of ftractureing appears easily in freezing process, and when concentration exceeded 5%, the whole viscosity of ice-cube was too big, and the later stage is because the ice-cube is cut by section equipment in the lump when cutting the sample section, and the ice-cube that the viscosity degree is too high can the adhesion on cutting the section, influences the operation of section equipment cutter and the fashioned quality of section image.
3. The introduction of the gelatin can also improve the freezing point of the embedding liquid and save the power consumption of the refrigeration equipment to a certain extent.
4. The addition of the pigment is beneficial to distinguishing the boundary between the internal target object and the surrounding ice block, and the smooth operation of the next step is ensured.
5. The active agent can ensure that each added component is in a suspension state in the embedding liquid, and the layering phenomenon after ice cake cutting is avoided.
Detailed Description
The present invention will be further described with reference to the following examples.
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The specimen (which can be an animal or a human body) is embedded to form a regular object, the regular object is placed in a slicing device (such as a milling machine) to be subjected to frozen slicing operation, and the image data (which can be a cross section, a longitudinal section or a sagittal section) of each slice is acquired and is subjected to 3D reconstruction to form a virtual digital image (such as a virtual digital human) for the fields of medical teaching, research and the like, so that specimen resources can be saved. In the embedding process, embedding liquid is required to be added into a box body with a specific shape to wrap a specimen, and a regular object is formed after freezing and is used for subsequent operations such as slicing, image acquisition and the like.
The ice blocks frozen by the existing embedding liquid are hard and brittle and are easy to crack in the processes of carrying and slicing the specimen; in the freezing process of embedding liquid, the composition of current embedding liquid is difficult for keeping the suspended state, has the phenomenon of subsiding in freezing process, causes the ice-cube fragility inconsistent after freezing, and the section of cutting then presents turbid layering phenomenon under the supernatant, can disturb follow-up to the precision of section image recognition, acquisition and 3D reconstruction. Meanwhile, the color difference between the color of the embedded embedding liquid ice block and the color of the boundary of the specimen is small, so that the boundary of the specimen is difficult to distinguish in the subsequent slice image acquisition and 3D reconstruction processes, and the width and height data of the section of the specimen are difficult to acquire.
Therefore, the following examples propose a specimen embedding fluid and a preparation method thereof, wherein water is used as a base fluid, gelatin, an active agent and a pigment are added in a set proportion, the formed embedding fluid is in a colloid state at 10 ℃ by utilizing the gelatin, and the brittleness is reduced after freezing; the active agent is utilized to enable the components in the embedding liquid to be in a suspension state, so that the brittleness is further reduced after freezing, and ice blocks cannot be layered; the pigment is used for distinguishing the boundary between the specimen in the embedding liquid and the ice block around the specimen.
In the following examples, the ingredients and the proportions of the embedding liquid added improve the overall hardness of the ice after freezing and forming, and at the same time, the ingredients are uniformly distributed to the maximum extent.
In the following embodiments, a method of adding a special pigment to the embedding liquid can ensure that the surface layer of the specimen is not polluted and the boundary can be better distinguished.
The first embodiment is as follows:
a specimen embedding fluid comprising: 3-5% of gelatin, 1-3% of active agent, pigment not exceeding 1.5 parts per million, and the balance of water.
The active agent is sodium dodecyl sulfate (C1)2H25SO4Na)。
The pigment includes blue methylene blue (CHN)3ClS) or red rhodamine B (C)28H31ClN2O3)。
The embedding liquid is jelly-shaped when cooled to 10 ℃.
Specifically, the method comprises the following steps:
the main component of the embedding liquid is water, and the main additive materials comprise: gelatin, active agents, pigments (colorants), and the like. The proportion of the added materials can be adjusted according to different requirements of environment and purposes.
For example, a human body specimen with a length of 1.7m is embedded, and the human body specimen is made of gelatin with a concentration of 3-5%, an active agent with a concentration of 1-3 per thousand, a pigment with a concentration of not more than 1.5 ten thousandths, and water as the rest.
The active agent generally used is sodium lauryl sulfate (C1)2H25SO4Na) as a white or yellowish powder, readily soluble in water. Commonly used pigments are: blue methylene blue (CHN)3ClS) and red rhodamine B (C)28H31ClN2O3) And is easily soluble in water.
The embedding fluid performance parameters in this example are shown in table 1.
Table 1: embedding fluid performance parameters
The addition of the gelatin can improve the viscosity of water, reduce the brittleness of ice cubes and ensure the stable later processing. Through a large number of experiments, the effect is best when the concentration is 3-5%.
When gelatin concentration is less than 3%, the whole fragility of ice-cube is too big, the phenomenon of ftractureing appears easily in freezing process, and when concentration exceeded 5%, the whole viscosity of ice-cube was too big, and the later stage is because the ice-cube is cut by section equipment in the lump when cutting the sample section, and the ice-cube that the viscosity degree is too high can the adhesion on cutting the section, influences the operation of section equipment cutter and the fashioned quality of section image.
The introduction of the gelatin can also improve the freezing point of the embedding liquid and save the power consumption of the refrigeration equipment to a certain extent.
The addition of a proper amount of pigment is beneficial to distinguishing the boundary of an internal target object and a surrounding ice block, and the smooth operation of the next step is ensured.
The active agent can ensure the suspension state of each main added component. Through experiments, the fluorescent agent is added into the embedding liquid, and under the irradiation of ultraviolet rays and the like, the Tyndall effect can be generated, which indicates that no obvious precipitation exists. In the process of freezing and forming, the consistency of the whole hardness is ensured as much as possible.
Example two:
the embodiment provides a preparation method for realizing the embedding solution, which comprises the following steps:
mixing gelatin with water according to the weight ratio of 1: 5 for at least 1 hour to obtain a gelatin solution, heating the gelatin solution and stirring to a gel state;
according to the following steps: 500 of active agent aqueous solution;
dropwise adding the gelatin solution in a gel state into the active agent aqueous solution, stirring to obtain a mixed solution, and heating the mixed solution to form a fluid state;
adding pigment into the fluid mixed solution, and cooling to normal temperature to form the prepared embedding solution.
The method comprises the following specific steps:
gelatin is mixed with water at 25 ℃ in a room temperature environment according to the ratio of 1: 5 for 1 hour to obtain the gelatin solution.
Heating the gelatin solution in water bath at 45 deg.C, and stirring to gel state.
At 25 ℃ in a room temperature environment, according to the following ratio of 1: 500 to prepare an aqueous solution of the active agent.
The gelatin solution in the gel state is added dropwise into the aqueous solution of the active agent, and the mixture is stirred to obtain a mixed solution.
The mixed solution was heated in a water bath at 45 ℃ to be fluid.
A coloring matter is added to the fluid mixed solution.
Cooling to normal temperature to form the prepared embedding liquid.
The addition of the gelatin can improve the viscosity of water, reduce the brittleness of ice cubes and ensure the stable later processing. Through a large number of experiments, the effect is best when the concentration is 3-5%.
When gelatin concentration is less than 3%, the whole fragility of ice-cube is too big, the phenomenon of ftractureing appears easily in freezing process, and when concentration exceeded 5%, the whole viscosity of ice-cube was too big, and the later stage is because the ice-cube is cut by section equipment in the lump when cutting the sample section, and the ice-cube that the viscosity degree is too high can the adhesion on cutting the section, influences the operation of section equipment cutter and the fashioned quality of section image.
The introduction of the gelatin can also improve the freezing point of the embedding liquid and save the power consumption of the refrigeration equipment to a certain extent.
The addition of a proper amount of pigment is beneficial to distinguishing the boundary of an internal target object and a surrounding ice block, and the smooth operation of the next step is ensured.
The active agent can ensure the suspension state of each main added component.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A specimen embedding liquid is characterized in that: comprises 3-5% of gelatin, 1-3% of active agent, no more than 0.15% of pigment, and the balance of water.
2. The specimen embedding fluid according to claim 1, wherein: the active agent is sodium dodecyl sulfate.
3. The specimen embedding fluid according to claim 1, wherein: the pigment comprises methylene blue or rhodamine B.
4. The specimen embedding fluid according to claim 1, wherein: the embedding liquid is in a jelly shape when being cooled to 10 ℃.
5. A process for the preparation of an embedding fluid according to any one of claims 1 to 4, comprising the steps of:
gelatin and water were mixed in a ratio of 1: 5, preparing a gelatin solution by soaking, heating and stirring the gelatin solution;
according to the following steps: 500 of active agent aqueous solution;
dropwise adding the gelatin solution into the active agent aqueous solution, stirring to obtain a mixed solution, and heating the mixed solution to form a fluid state;
adding pigment into the fluid mixed solution, and cooling to normal temperature to form the prepared embedding solution.
6. The method of claim 5, wherein: the gelatin is soaked in water for at least 1 hour.
7. The method of claim 5, wherein: the gelatin is soaked with water at room temperature, and the aqueous solution of the active agent is prepared at room temperature.
8. The method of claim 5, wherein: the gelatin solution is heated and stirred to a gel state and then added dropwise to an aqueous solution of the active agent.
9. The method of claim 5, wherein: and the gelatin solution and the mixed solution are heated in a water bath manner.
10. The method of claim 5, wherein: the heating temperature of the gelatin solution and the mixed solution is not lower than 45 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111470891.3A CN114097766A (en) | 2021-12-03 | 2021-12-03 | Specimen embedding liquid and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111470891.3A CN114097766A (en) | 2021-12-03 | 2021-12-03 | Specimen embedding liquid and preparation method thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170160174A1 (en) * | 2014-06-27 | 2017-06-08 | National University Corporation Nagoya University | Embedding medium for specimen preparation, method for preparing curable base material nonpenetrating specimen, method for preparing curable base material penetrating specimen, curable base material nonpenetrating specimen, thin slice performance improver for frozen embedding medium, and frozen embedding medium |
| CN108225876A (en) * | 2017-12-28 | 2018-06-29 | 广州大学 | A kind of sample embedding method |
| CN108575983A (en) * | 2018-04-24 | 2018-09-28 | 深圳大学 | A kind of meiofauna cutting frozen embedding method |
| CN113702148A (en) * | 2021-08-27 | 2021-11-26 | 华中科技大学同济医学院附属协和医院 | Freezing embedding agent and application thereof in frozen section |
-
2021
- 2021-12-03 CN CN202111470891.3A patent/CN114097766A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170160174A1 (en) * | 2014-06-27 | 2017-06-08 | National University Corporation Nagoya University | Embedding medium for specimen preparation, method for preparing curable base material nonpenetrating specimen, method for preparing curable base material penetrating specimen, curable base material nonpenetrating specimen, thin slice performance improver for frozen embedding medium, and frozen embedding medium |
| CN108225876A (en) * | 2017-12-28 | 2018-06-29 | 广州大学 | A kind of sample embedding method |
| CN108575983A (en) * | 2018-04-24 | 2018-09-28 | 深圳大学 | A kind of meiofauna cutting frozen embedding method |
| CN113702148A (en) * | 2021-08-27 | 2021-11-26 | 华中科技大学同济医学院附属协和医院 | Freezing embedding agent and application thereof in frozen section |
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Application publication date: 20220301 |