CN111413184A - Staining device and biological tissue marking apparatus - Google Patents
Staining device and biological tissue marking apparatus Download PDFInfo
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- CN111413184A CN111413184A CN202010369157.7A CN202010369157A CN111413184A CN 111413184 A CN111413184 A CN 111413184A CN 202010369157 A CN202010369157 A CN 202010369157A CN 111413184 A CN111413184 A CN 111413184A
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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
- G01N1/31—Apparatus therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The invention provides a staining device and biological tissue marking equipment which are simple in system, small in size, easy to realize, low in cost and good in marking effect. The dyeing apparatus provided by the invention is electrified to mark biological tissues, and comprises: a dyeing chamber body having a receiving cavity; the antibody groove is detachably arranged in the containing cavity, and two lateral surfaces of the antibody groove are symmetrically provided with semipermeable membranes communicated with the dyeing chamber main body and used for containing an antibody solution serving as a dyeing solution; a sample tank detachably provided in the antibody tank, and having a through hole for accommodating the biological tissue at a position corresponding to the semipermeable membrane; and two electrode plates, parallel and symmetry setting are in the both sides in antibody groove, the lower extreme with the external power supply of dyeing chamber main part is connected, is used for to the encirclement biological tissue the antibody circular telegram, and right biological tissue marks, wherein, the molecular cut-off volume of pellicle is 6 ~ 100kDa, the material in antibody groove is insulating material.
Description
Technical Field
The invention relates to the technical field of biological experiments, in particular to a dyeing device and biological tissue marking equipment.
Background
Although staining biological tissue is a common working method in the biological and medical fields, staining thick tissue (thickness in the order of millimeters or more) is one of the difficulties in the life science field. Traditional passive diffusion tissue labeling methods based on staining molecules (usually antibodies) typically require weeks to complete a thick tissue stain. The active diffusion method based on external force (such as electric field) driving can accelerate the transport speed of the staining molecules in the tissues and greatly reduce the staining time. However, the existing immune marker accelerates the diffusion of antibody molecules in dense tissues based on the random electric field transport principle, so that the electric field strength needs to be more than 1151V/m, and in order to meet the condition, the voltage of the marker is generally more than 100V, so that the device is not only complicated, but also large in volume and generates large heat. At a voltage of 100V, the current is generally above 1A, the total power is above 100W, and therefore a refrigeration system is necessary, otherwise the temperature of the system quickly exceeds the temperature limit (namely 37 ℃) which can be borne by the sample, so that the cost of the whole immunity marking instrument is increased. There is an urgent need for an immunolabeling device that is small in size and low in cost.
Disclosure of Invention
Therefore, in order to overcome the disadvantages of the prior art, a staining apparatus and a biological tissue marking device are provided, which have the advantages of simple system, small volume, easy implementation, low cost and good marking effect.
In order to achieve the above object, the present invention provides a staining apparatus that marks a biological tissue by applying a current thereto, comprising: a dyeing chamber body having a receiving cavity; the antibody groove is detachably arranged in the containing cavity, and two lateral surfaces of the antibody groove are symmetrically provided with semipermeable membranes communicated with the dyeing chamber main body and used for containing an antibody solution serving as a dyeing solution; a sample tank detachably provided in the antibody tank, and having a through hole for accommodating the biological tissue at a position corresponding to the semipermeable membrane; and two electrode plates, parallel and symmetry setting are in the both sides in antibody groove, the lower extreme with the external power supply of dyeing chamber main part is connected, is used for to the encirclement biological tissue the antibody circular telegram, and right biological tissue marks, wherein, the molecular cut-off volume of pellicle is 6 ~ 100kDa, the material in antibody groove is insulating material.
In one embodiment, a partition for installing the antibody slot is disposed in the receiving cavity of the dyeing chamber body.
In one embodiment, the electrode plate is a sheet or conductive sheet wound with conductive filaments.
In one embodiment, the electrode plate is a platinum sheet, a gold sheet, or a graphite plate.
In one embodiment, the semi-permeable membrane ranges in size from 5mm by 5mm to 50mm by 50 mm.
The present invention also provides a biological tissue marking apparatus comprising: a staining device for marking biological tissue; and the electrophoresis device is electrically connected with the dyeing device, is connected with an external power supply and is used for providing current during marking, wherein the dyeing device is the dyeing device.
In one embodiment, the current control device includes: the shell is provided with a display screen for receiving dyeing data; the dyeing chamber power supply is detachably and electrically connected with the dyeing device, is used for electrifying the antibody in the dyeing device, and is provided with a power supply input port communicated with an external electrophoresis power supply and a current output port connected with the dyeing device; the control chip is electrically connected with the display screen and the current output port and controls the current value and the current direction output by the current output port according to the dyeing data; and the working power supply input port is communicated with an external working power supply and is used for supplying power to the display screen and the control chip.
Compared with the prior art, the invention has the advantages that: based on the principle of low electric field assisted antibody molecule diffusion, an external force is superposed to accelerate the diffusion of antibody molecules in compact tissues while antibody molecules are freely diffused through an external weak electrostatic field (<600V/m), so that the working voltage is less than 30V, in order to ensure that all current can only pass through biological tissues, the periphery of the biological tissues is completely blocked by using a non-conductive resin material, and under the condition of high resistance of a sample, the working current is only in mA magnitude, so that the power is less than 1W magnitude, the generated heat can be almost ignored, and no additional cooling system is required to be added. The whole device is simple, small in size, easy to realize, low in cost and good in marking effect, and can realize rapid and uniform immunofluorescence marking of thick (the thickness is more than millimeter magnitude) biological tissues (animals, clinical samples and plants).
Drawings
FIG. 1 is a schematic diagram of the structure of a biological tissue marker device in an embodiment of the invention;
FIG. 2 is a schematic view showing the structure of a dyeing apparatus in an embodiment of the present invention;
FIG. 3 is a disassembled view of a dyeing apparatus in an embodiment of the present invention;
FIG. 4 is a graph showing the effect of staining of a biological tissue marking device in an embodiment of the present invention;
FIG. 5 is a graph showing the dyeing effect of the conventional apparatus.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
As shown in fig. 1, in one embodiment of the present invention, biological tissue marking apparatus 100 includes a staining device 200 and an electrophoresis device 300.
The staining apparatus 200 is used to fix and mark biological tissues. The biological tissue can be animal tissue, clinical sample tissue and plant tissue, the thickness is from millimeter magnitude to centimeter magnitude, and the biological tissue can be a sample after transparentization treatment or a sample without transparentization. As shown in fig. 2 and 3, the staining apparatus 200 includes a staining chamber body 10, an antibody well 20, a sample well 30, and an electrode plate 40.
The dyeing chamber body 10 has a receiving cavity. The dyeing chamber body 10 has a magnetic base 11 and a cylindrical housing 12. The staining chamber body 10 may be made by 3D printing or non-metal processing, using non-metallic materials that are corrosion resistant and not prone to wear, typically including photosensitive resins, plexiglass, glass, plastics, etc. The dyeing chamber body may have a circular or square receiving cavity. In this embodiment, the accommodating cavity may be a cylinder with a diameter of 40-120mm and a height of 40-120mm, and a partition board of the antibody slot 20 may be disposed in the accommodating cavity, and the partition board divides a rectangular area from the accommodating cavity, wherein the rectangular area is an electrophoresis immune labeling area and has a side length of 20-50 mm. The accommodating cavity is divided into two parts serving as buffer areas by a rectangular area. In one embodiment, the containment chamber is a cylindrical chamber having a diameter of about 78mm and a height of about 80mm, and the rectangular area is 30mm by 36 mm. The cup body design of the arc-shaped containing cavity is easier for operators to hold, is not easy to drop and simultaneously improves the attractiveness.
The antibody tank 20 is detachably provided in the holding chamber and holds an antibody solution as a staining solution. The antibody groove is a rectangular square groove. The arc-shaped handle is arranged on the short side of the rectangular square groove, so that an operator can conveniently take the antibody groove 20 out of the accommodating cavity. The material of the antibody groove is an insulating material, and the resistivity of the insulating material is more than 109Omega.m. The side surfaces of the two long edges are symmetrically provided with through holes which can be communicated with the dyeing chamber main body, the through holes are provided with semipermeable membranes, the semipermeable membranes are made of regenerated fibers or other fiber materials, and the molecular interception amount of the semipermeable membranes is 6-100 kDa. The size of the semi-permeable membrane ranges from 5mm by 5mm to 50mm by 50 mm. In one embodiment, the semi-permeable membrane has dimensions of 15mm by 25 mm.
The sample well 30 is detachably provided in the antibody well 20, and a through-hole for accommodating a biological tissue is provided at a position corresponding to the semipermeable membrane. The diameter of the through-going hole may be 15-60mm, in one embodiment the diameter of the through-going hole is 33 mm. The sample well 30 is provided with a handle along the long side for easy access. The biological tissue may be fixed in the through-holes by epoxy glue, agarose, or the like.
The electrode plates 40 are provided in two, parallel and symmetrical positions on both sides of the antibody slot. The lower end of the electrode plate 40 is connected to an external power source of the staining chamber main body, and is used for energizing the antibody surrounding the biological tissue and labeling the biological tissue. In one embodiment, the electrode plate 40 is a sheet or conductive sheet wound with conductive filaments. The electrode plate 40 may be a metal or nonmetal material that can be used as an electrode, such as a platinum plate, a gold plate, or a graphite plate. The electrode plate 40 may be a metal plate to which a specific electrode is fixed. The spacing between the two electrode plates 40 may be 15-60mm, and in one embodiment, the spacing is 33 mm. A small hole opening is provided at a predetermined position of the dyeing chamber main body, and the lower end of the electrode plate 40 passes through the small hole opening to be electrically connected to a non-wired connector of the external electrophoresis apparatus 300. Waterproof silicon rubber is arranged at the opening of the small hole, so that the dyeing liquid is prevented from leaking into a circuit.
The electrophoresis device 300 is electrically connected to the dyeing device 200 for supplying a current at the time of marking. Electrophoresis apparatus 300 includes housing 301, staining chamber power supply 302, control chip 303, and operating power input port 304.
The shell 301 is provided with a display screen for receiving dyeing data, the shell 301 can be made of metal materials or plastic materials or resin materials with certain strength, the shell has a supporting structure and has the function of protecting internal circuits, a front panel of the shell 301 can be arranged to form an acute angle with the horizontal plane, in one embodiment, the front panel is arranged to be inclined to the horizontal plane by 30 degrees, and accordingly, an operator can conveniently read numbers on the front panel and input the numbers, the size range of the shell 301 can be 10-30 cm in length, 10-30 cm in width and 2-10 cm in height, in one embodiment, the overall size is × in length, × in width and 12 x 13 x 2.5cm in height, and the shell is small, small and light and convenient to use in a conventional biological laboratory and a clinical examination laboratory.
The display screen may include membrane keys, a digital display and a control panel to enable interaction of the instrument operator with the dye meter. The film key is a key with an input function, which is made of materials such as plastics or metal. The digital display displays key parameters of the system including run time, remaining time, etc. Parameters related to the dyeing data can be input through keys, so that the operation and the stop of the instrument can be controlled. The control panel is fixed on the front panel of the shell 301 with a certain inclination angle, so that the operator can conveniently check and operate in a standing or sitting state.
The display screen may also include status indicator lights. The status indicator light may indicate the status of the instrument, including operational and non-operational, etc. When the test device is in an operating state, the number of the display screen enters countdown, and a prompt tone is sent to remind an operator when the remaining time is less than 1-5 minutes or the test device stops accidentally, so that corresponding operation is carried out to ensure that the test is carried out smoothly. The film key, the digital display, the status indicator light and the control panel are electrically connected.
The staining chamber power supply 302 communicates with an external power supply, and has a power input port 3021 provided on the rear panel of the housing 301 and a current output port 3022 connected to the staining apparatus.
The power input port 3021 may be connected to a constant current source or a 220V ac circuit by connecting a power adapter, and is provided with a power switch. All joints are firmly connected, an insulating protective cover is provided, and electric leakage can be effectively prevented.
The current output port 3022 is electrically connected to the dyeing apparatus. The current output port 3022 is provided at the top end of the housing 301, and is connected to a connection terminal at the bottom of the dyeing apparatus 200. The current output port 3022 may be connected to the dyeing apparatus 200 and the electrophoresis apparatus 300 by any one of a magnetic connector, a banana head, and a socket head. The connector is conducted, so that the ampere-magnitude current can be stably maintained, at least ten thousand plugging times can be kept, and the service life of the instrument is not influenced.
The control chip 303 is electrically connected to the display screen, the power input port 3021, and the current output port 3022, and controls a current value and a current direction output from the current output port 3022 according to the coloring data. The control chip 303 is fixed to a bottom plate inside the housing 301. In the present embodiment, the control chip 303 is a Printed Circuit Board (PCB). The PCB circuit board can realize automatic current control of the dyeing process. The total time of the power-on can be input through the display screen according to the experiment requirement. The control chip 303 automatically controls the electrophoresis power supply to supply a constant current to the dyeing apparatus 200 according to the total time. When the running time reaches exactly half of the total time, the control chip 303 automatically reverses the current and keeps the constant current until the end of the experiment. The two times of immune labeling of the experimental sample is realized through the current reversal, and the uniformity of the immune labeling is ensured. The control chip 303 can memorize the time input by the operator last time and display the time on the display screen, so that the operator can conveniently repeat experiments or perform other operations. In the experimental process, an operator can control the running, the pause, the continuation and the stop of the instrument through keys of the display screen. Control chip 303 can detect whether circuit connection is normal before the operation, if dyeing apparatus does not put the condition that correctly leads to circuit connection unusual, can remind operating personnel to place dyeing apparatus again automatically.
The operating power input port 304 communicates with an external operating power source for providing power to the display screen of the housing 301 and the control chip 303. The operating power input port 304 may be a power adapter that is connected to 220V ac and provides a stable operating voltage of 12-24V.
The use flow of the dyeing apparatus 200 is:
fixing the experimental sample (thick biological tissue) in the circular cavity of the sample groove 30 by a fixing agent; then the sample groove 30 is arranged at the corresponding position of the antibody groove 20; then, an antibody diluent (dilution ratio 1: 200-1: 10000 times) may be added to both sides of the sample well 30 of the antibody well 20 until it passes over the semi-permeable membrane of the antibody well 20; the antibody tank 20 is placed at a position corresponding to the dyeing chamber main body 10, and the dyeing solution is added to the remaining space of the dyeing chamber until the electrode plate is submerged.
The use flow of the biological tissue marking device is as follows:
placing the staining apparatus 200 on top of the electrophoresis apparatus 300; connecting an external power supply to a working power supply input port 304 of a rear panel of the control box, and turning on a power switch of the instrument to enable the instrument to be in a state to be operated;
inputting the total duration of the experiment through a display screen; after the setting is finished, clicking an operation button, automatically calculating the time before and after the current is reversed by the control chip 303, switching a state indicator lamp and a forward conducting circuit, and automatically switching to reverse conducting after the operation time in one direction (defined as forward direction) is finished until the experiment is finished;
when the experiment is finished, a prompt tone is generated, and an operator can take out the sample, or replace the kit for cleaning, or replace the antibody for immunological marking again.
The dyeing device and the biological tissue marking equipment are based on the principle that the low electric field assists the diffusion of antibody molecules, and an external force is superposed to accelerate the diffusion of the antibody molecules in compact tissues while the antibody molecules are freely diffused through an external weak electrostatic field (<600V/m), so that the working voltage is less than 30V, in order to ensure that all current can only pass through the biological tissues, the periphery of the biological tissues is completely blocked by using a non-conductive resin material, and under the condition of high resistance of a sample, the working current is only in the mA magnitude, so that the power cannot exceed 1W, the generated heat can be almost ignored, and no additional cooling system is required to be added. The whole device is simple, small in size, easy to realize, low in cost and good in marking effect, and can realize rapid and uniform immunofluorescence marking of thick (the thickness is more than millimeter magnitude) biological tissues (animals, clinical samples and plants).
In one embodiment, the dyeing chamber main body is further provided with a cover, and the cover can effectively prevent the dyeing solution from overflowing. The cover is provided with a rectangular liquid inlet and outlet with the side length of 5-40mm, and the rectangular liquid inlet and outlet can facilitate liquid adding and cleaning of operators. In one embodiment, the rectangular inlet and outlet may be 15mm by 25mm in size.
For example, the Anti-Histone H3 antibody with Alexa-647 fluorescent molecule can be used to stain and label hyalinized brain tissue with a thickness of 2mm as follows:
firstly, carrying out transparent treatment on mouse brain (C57B L/6) tissues with the thickness of 2mm by using a standard C L ARITY transparentizing method;
fixing the sample in the sample groove 30 by using epoxy resin glue, and placing the sample groove 30 at the corresponding position of the antibody groove 20;
adding an Anti-histone H3 antibody solution diluted at a ratio of 1:200 into the antibody tank 20 until the antibody tank 20 is submerged in the semipermeable membrane;
placing the antibody groove 20 at the corresponding position of the dyeing chamber main body 10, adding 100m of the dyeing solution L into the residual space of the dyeing chamber, and submerging the electrode plate;
placing the staining apparatus 200 on top of the electrophoresis apparatus 300;
switching on a power supply of the dyeing device, inputting dyeing time of 3 hours, and starting the dyeing device;
after dyeing is finished, taking out a sample, placing the sample into a special sample groove for elution, and replacing the antibody solution with a special buffer solution for elution;
and putting the sample into a staining chamber, starting a power supply and inputting for 3 hours, and starting a staining device to elute the antibody.
In the present experiment, the staining time was 3 hours, the elution time was 3 hours, the voltage output was 20V, the current output was 20mA, the power output was only 0.4W, the actual amount of antibody (concentration × vol) was only 0.6 μ g, the increase in system temperature during the entire staining process did not exceed 1 ℃.
Under the same conditions, FIG. 5 is a fluorescence image of a cross section of a brain slice after a mouse brain tissue (2mm thickness) is stained with a staining apparatus using a passive staining method for the same staining time and antibody concentration. The white scale of fig. 4 and 5 is 200 μm. As shown in fig. 5, the staining apparatus only marks a very thin area (<200 μm) of the surface, whereas the time required for immunolabeling needs to be extended more than 10 times to achieve the staining effect as in fig. 4.
Therefore, according to the same tissue thickness calculation, the antibody dosage of the device can be 1/6 of the traditional passive staining device and 1/3 of a random electric field transportation method, and the device can greatly save the cost of the immune marker while ensuring the quality of immune staining.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention. The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A staining apparatus that is energized to mark biological tissue, comprising:
a dyeing chamber body having a receiving cavity;
the antibody groove is detachably arranged in the containing cavity, and two lateral surfaces of the antibody groove are symmetrically provided with semipermeable membranes communicated with the dyeing chamber main body and used for containing an antibody solution serving as a dyeing solution;
a sample tank detachably provided in the antibody tank, and having a through hole for accommodating the biological tissue at a position corresponding to the semipermeable membrane; and
two electrode plates which are arranged at two sides of the antibody groove in parallel and symmetrically, the lower ends of the two electrode plates are connected with an external power supply of the staining chamber main body, and the two electrode plates are used for electrifying the antibody surrounding the biological tissue and marking the biological tissue,
wherein the molecular interception amount of the semipermeable membrane is 6-100 kDa, and the material of the antibody groove is an insulating material.
2. The staining apparatus of claim 1, wherein a partition for installing the antibody slot is provided in the receiving cavity of the staining chamber body.
3. The dyeing apparatus according to claim 1, characterized in that said electrode plates are sheets of wound conductive filaments or conductive strips.
4. The dyeing apparatus according to claim 1, characterized in that said electrode plate is a platinum sheet or a gold sheet.
5. The staining apparatus of claim 1, wherein the semi-permeable membrane has a size ranging from 5mm by 5mm to 50mm by 50 mm.
6. A biological tissue marking apparatus, comprising:
a staining device for marking biological tissue; and
an electrophoresis device electrically connected with the dyeing device and connected with an external power supply and used for providing current during marking,
wherein the dyeing device is the dyeing device as claimed in any one of claims 1 to 5.
7. A biological tissue marking apparatus as claimed in claim 6 wherein the electrophoresis means comprises:
the shell is provided with a display screen for receiving dyeing data;
the dyeing chamber power supply is detachably and electrically connected with the dyeing device, is used for electrifying the antibody in the dyeing device, and is provided with a power supply input port communicated with an external electrophoresis power supply and a current output port connected with the dyeing device;
the control chip is electrically connected with the display screen and the current output port and controls the current value and the current direction output by the current output port according to the dyeing data; and
and the working power supply input port is communicated with an external working power supply and is used for providing power for the display screen and the control chip.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010369157.7A CN111413184A (en) | 2020-05-02 | 2020-05-02 | Staining device and biological tissue marking apparatus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111982638A (en) * | 2020-08-20 | 2020-11-24 | 上海诒福科技有限公司 | Biological tissue staining method, biological tissue staining apparatus, computer device, and storage medium |
| CN113049804A (en) * | 2021-03-17 | 2021-06-29 | 上海交通大学 | Method for rapidly marking biological tissues |
| CN114088501A (en) * | 2021-11-12 | 2022-02-25 | 南通大学 | Chip device for in-situ tissue staining and decoloring and use method |
-
2020
- 2020-05-02 CN CN202010369157.7A patent/CN111413184A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111982638A (en) * | 2020-08-20 | 2020-11-24 | 上海诒福科技有限公司 | Biological tissue staining method, biological tissue staining apparatus, computer device, and storage medium |
| CN111982638B (en) * | 2020-08-20 | 2023-11-10 | 诒福生物科技南通有限公司 | Biological tissue staining method, apparatus, computer device, and storage medium |
| CN113049804A (en) * | 2021-03-17 | 2021-06-29 | 上海交通大学 | Method for rapidly marking biological tissues |
| CN114088501A (en) * | 2021-11-12 | 2022-02-25 | 南通大学 | Chip device for in-situ tissue staining and decoloring and use method |
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