CN108572099B - Method and device for realizing rapid staining of biological tissues - Google Patents
Method and device for realizing rapid staining of biological tissues Download PDFInfo
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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
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Abstract
The device for realizing the rapid dyeing of the biological tissues comprises a dyeing box, wherein the dyeing box is internally provided with 1-100 electrophoresis cavity placing grooves; the electrophoresis cavity holding groove is provided with an electrophoresis cavity, a temperature sensor, an ion concentration sensor, a pH sensor, a current sensor and a flow velocity sensor. The device provided by the invention can effectively improve the dyeing efficiency, can greatly reduce the dyeing time, obviously improves the dyeing quality, and has the advantages of simplicity, strong universality, easiness in assembly and low cost.
Description
Technical Field
The invention relates to the field of biological devices, in particular to a method and a device for realizing rapid staining of biological tissues.
Background
The development and the gradual maturation of tissue clearing techniques in recent years have made it possible to obtain high resolution three-dimensional structures of intact biological tissues. However, when applying tissue clearing techniques to study the three-dimensional high resolution structure of an intact tissue or organ, the time required to stain biological tissue is often long.
For example, a single immunofluorescence staining of the whole brain of a cleared mouse often takes several weeks. The bottleneck of overlong dyeing time greatly limits the application of the tissue transparentization technology to the research of three-dimensional structures of tissues in different physiological states.
The main reason for the lengthy staining time is that the molecular probes for labeling the tissue, especially antibodies, take a long time to reach the interior of the tissue from the surface of the intact tissue in a passive diffusion manner.
In summary, there is a need in the art to develop a device and a method for rapid staining of biological tissue, which is convenient to use and simple in structure.
Disclosure of Invention
The invention aims to develop a device and a method which are convenient to use, simple in structure and capable of realizing rapid staining of biological tissues.
The invention provides a device for realizing quick staining of biological tissues in a first aspect, which comprises a staining box;
wherein, the dyeing box is internally provided with 1 to 100 electrophoresis chamber placing grooves;
the electrophoresis cavity holding groove is provided with an electrophoresis cavity, a temperature sensor, an ion concentration sensor, a pH sensor, a current sensor and a flow velocity sensor;
the outer side of the notch of the electrophoresis cavity holding groove is provided with a positive electrode interface and a negative electrode interface;
the electrophoresis chamber is provided with a positive electrode, a negative electrode, a sample chamber fixing frame and a sample chamber.
In another preferred embodiment, the device is provided with a control system, and the control system comprises a control panel, a temperature control module, a hydraulic control module, a current module, a power switch, a time control module and a pH control module;
the temperature control module, the hydraulic control module and the current module are positioned below an electrophoresis cavity placing groove in the device;
the temperature control module is connected with the temperature sensor;
the hydraulic control module is connected with the flow velocity sensor, the ion concentration sensor and the pH sensor;
the current module is connected with the power switch and the current sensor.
In another preferred example, the distance between the positive electrode and the cavity wall is 0.01-0.5cm, and the distance between the negative electrode and the cavity wall is 0.01-0.5 cm.
In another preferred example, the positive electrode comprises an electrode material and a fixed electrode material:
the material of the fixed electrode is an insulating material, and the material of the fixed electrode is selected from the following group: a glass plate, a plastic plate, a ceramic plate, or a combination thereof;
the electrode material is a conductive material, and is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof;
in another preferred example, the material of the electrode is platinum metal.
In another preferred example, the negative electrode comprises an electrode material and a material for fixing the electrode;
the fixed electrode material is an insulating material and is selected from the following group: a glass plate, a plastic plate, a ceramic plate, or a combination thereof;
the electrode material is a conductive material, and is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof.
In another preferred example, the material of the electrode is platinum metal.
In another preferred example, the control panel has a temperature adjustment panel, an ion concentration adjustment panel, a pH adjustment panel, a current adjustment panel, a time adjustment panel, and a flow rate control panel.
In another preferred embodiment, the sample chamber comprises: the top cover, the inner ring, the bottom cover, the first dialysis membrane and the second dialysis membrane;
wherein, the inner ring includes: hollow column of upper agarose, sample, hollow column of lower agarose
Wherein, the sample chamber include from one end to the other end in proper order: top cap, first dialysis membrane, inner ring, bottom, second dialysis membrane.
In another preferred embodiment, the first dialysis membrane has a molecular weight cut-off of 6-500kDa and the second dialysis membrane has a molecular weight cut-off of 6-500 kDa.
A second aspect of the invention provides a method of the apparatus of the first aspect of the invention, the method comprising the steps of:
(I) placing an electrophoresis cavity containing buffer solution in an electrophoresis cavity placing groove, placing a sample cavity in a sample cavity fixing frame, and adjusting the current between a positive electrode and a negative electrode; wherein the buffer solution has an ion concentration of 0.1-10M, pH of 1-13 and a current intensity of 0-300 mA;
(II) dyeing, wherein the dyeing time is 30-48 hours.
In another preferred embodiment, the method further comprises the steps of: and (3) eluting, wherein in the step of eluting, the ion concentration of the buffer solution is 0.1-10M, pH and is 1-13, the current intensity is 0-500mA, and the elution time is 30min-24 hours.
In another preferred example, the current ranges from 0.1 to 300mA, and the direction of the current is changed alternately.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a schematic view of the apparatus.
FIG. 2 is a schematic diagram of the relationship of the control system to the modules.
Fig. 3 is a top view of the device.
Figure 4 is a schematic view of the interior of an electrophoresis chamber.
Figure 5 is a schematic view of a sample chamber.
Fig. 6 is the imaging result of example 1.
Fig. 7 is the imaging result of example 2.
Fig. 8 is the imaging result of example 3.
In the figure, the respective designations are as follows:
1: a control panel;
2: a power switch;
3: an electrophoresis chamber placement groove;
4: positive and negative electrode interfaces;
5: a hydraulic control module;
6: a temperature control module;
11: a sample chamber;
12: a positive electrode;
15: an electrophoresis chamber;
16: a negative electrode;
111: a top cover;
112: an inner ring;
113: a bottom cover;
114: a sample;
115: an upper agarose hollow cylinder;
116: a lower agarose hollow cylinder;
117: a second dialysis membrane;
118: a first dialysis membrane.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies for a long time, and unexpectedly found that the use of a small current whose electric field direction is alternately changed can accelerate the entry of a molecular probe into a tissue, prevent the tissue sample from being damaged by a large amount of joule heat, greatly reduce the dyeing time, and significantly improve the dyeing quality.
Term(s) for
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.
As used herein, the terms "device for achieving rapid staining of biological tissue", "device for achieving rapid staining of biological tissue" or "device" are used interchangeably and refer to a device for achieving rapid staining of biological tissue as described in the first aspect of the present invention.
Device for realizing quick dyeing of biological tissues
An apparatus for achieving rapid staining of biological tissue comprising: and (4) a dyeing box.
Wherein, the dyeing box is internally provided with 1 to 100 electrophoresis chamber placing grooves 3;
the electrophoresis cavity holding groove is provided with an electrophoresis cavity 15, a temperature sensor, an ion concentration sensor, a pH sensor, a current sensor and a flow velocity sensor.
And the outer side of the notch of the electrophoresis cavity holding groove is provided with a positive electrode interface and a negative electrode interface 4.
The electrophoresis chamber is provided with a positive electrode 12, a negative electrode 16, a sample chamber fixing frame and a sample chamber 11.
In another preferred example, the distance between the positive electrode and the cavity wall is 0.01-0.5cm, and the distance between the negative electrode and the cavity wall is 0.01-0.5 cm.
In another preferred example, the positive electrode comprises an electrode material and a fixed electrode material:
the material of the fixed electrode is an insulating material, and the material of the fixed electrode is selected from the following group: a glass plate, a plastic plate, a ceramic plate, or a combination thereof.
The electrode material is a conductive material, and is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof.
In another preferred example, the material of the electrode is platinum metal.
In another preferred example, the negative electrode comprises an electrode material and a fixed electrode material.
The fixed electrode material is an insulating material and is selected from the following group: a glass plate, a plastic plate, a ceramic plate, or a combination thereof.
The electrode material is a conductive material, and is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof.
In another preferred example, the material of the electrode is platinum metal.
The device is provided with a control system, wherein the control system comprises a control panel, a temperature control module, a hydraulic control module, a current module, a power switch, a time control module and a pH control module;
the temperature control module, the hydraulic control module and the current module are positioned below an electrophoresis cavity placing groove in the device;
the temperature control module is connected with the temperature sensor;
the hydraulic control module is connected with the flow velocity sensor, the ion concentration sensor and the pH sensor;
the current module is connected with the power switch and the current sensor.
In another preferred embodiment, the control panel has a temperature adjustment panel, an ion concentration adjustment panel, a pH adjustment panel, a current adjustment panel, a time adjustment panel, and a flow rate control panel.
The sample cavity of the electrophoresis cavity of the device for quickly staining the biological tissues comprises: a top cover 111, an inner ring 112, a bottom cover 113, an upper agarose hollow column 115, a lower agarose hollow column 116, a second dialysis membrane 117, and a first dialysis membrane 118, wherein the first dialysis membrane has a molecular weight cut-off of 6-500kDa, and the second dialysis membrane has a molecular weight cut-off of 6-500 kDa.
Wherein, the inner ring is inside to include: hollow cylinder of agarose at upper part, sample, hollow cylinder of agarose at lower part
Wherein, the sample chamber include from one end to the other end in proper order: top cap, first dialysis membrane, inner ring, bottom, second dialysis membrane.
Method of using a device
A method of using a device comprising the steps of:
(I) placing an electrophoresis cavity containing buffer solution in an electrophoresis cavity placing groove, placing a sample cavity in a sample cavity fixing frame, and adjusting the current between a positive electrode and a negative electrode; wherein the buffer solution has an ion concentration of 0.1-10M, pH of 1-13 and a current intensity of 0-300 mA;
(II) dyeing for 30min-48 hrs.
In another preferred embodiment, the method further comprises the steps of: eluting, wherein in the elution step, the ion concentration of the buffer solution is 0.1-10M, pH and is 1-13, the current intensity is 0-500mA, and the elution time is 30min-24 hours.
In another preferred embodiment, the direction of the electric field of the current is alternating.
The invention has the advantages that:
1. in favor of molecular probes, such as: the antibody molecules and the nucleic acid probe can be rapidly diffused in the tissue, so that the time for the probe to penetrate into the tissue can be greatly reduced, and the tissue can be rapidly dyed.
2. The small current is adopted, so that the molecular probe can rapidly enter the tissue, and the damage of a large amount of generated joule heat and electric field force to the sample can be avoided.
3. The direction of the small current electric field is changed alternately, the probability that antibody molecules are trapped in compact tissues is reduced, and the efficiency and the quality of staining are improved
4. The molecular probe solution is filled into both sides of the dyed sample, and in the dyeing process, the molecular probes can enter tissues from both sides, so that the dyeing time can be shortened, and the dyeing quality can be improved
5. The probe molecules which are not specifically adhered can be completely eluted by the action of the electric field force by the electric field elution, so that the background signal is greatly reduced, the dyeing is improved, and the imaging quality is improved
6. The device is simple, easy to assemble and low in cost.
Example 1
Testing of the device of the invention with IgG antibodies
(a) Assembled sample cavity
According to the sample chamber from one end to the other end in proper order including: the sample cavity is assembled by sequentially arranging the top cover, the first dialysis membrane, the inner ring, the bottom cover and the second dialysis membrane, wherein the interior of the sample cavity comprises an upper agarose hollow cylinder, a sample and a lower agarose hollow cylinder, and the first dialysis membrane and the second dialysis membrane need to remove protective glycerol attached to the surfaces by deionized water.
Wherein the sample is a brain slice of adult mouse (C57BL/6) with thickness of 1mm and made transparent by passive Clarity method
The assembling of the sample chamber comprises the steps of:
(a-1) adding an antibody to a hollow portion of a lower agarose hollow cylinder;
(a-2) placing the sample in a hollow portion of a lower agarose hollow cylinder and injecting 8% agarose around the sample;
(a-3) after the agarose is solidified, the upper agarose hollow cylinder is mounted, and the hollow portion of the upper agarose cylinder is filled with the antibody solution.
(b) Dyeing, wherein the dyeing comprises the following steps:
(b-1) electrophoretic fluid was used in the following ratio of 1: preparing an Anti-GFP-Alexa555) antibody solution at a ratio of 200;
(b-2) after the sample cavity is assembled, putting the sample cavity into an electrophoresis tank, fixing the sample cavity by using blue butyl rubber, adding a buffer solution, and covering the buffer solution with the sample cavity;
and (b-3) turning on a power supply, setting the current to be 25mA, running for 15min for electrophoresis, and then exchanging the current directions for positive and negative twice respectively for 1h, wherein the buffer solution is a sodium borate buffer solution, and the concentration of the buffer solution is 0.1mol/L, pH to be 8.5.
(c) Fluorescence imaging detection of marking effects
Cutting off the power supply, opening the top cover and the bottom cover, taking out the sample from the inner ring, cutting off excessive agarose, stripping off the sample, and imaging by a laser scanning confocal microscope (A1Si, Nikon) with the imaging multiple of 10X.
Imaging results
The imaging result is shown in fig. 6, and it can be seen that the transparent brain slice tissue with the thickness of 1mm is filled with the antibody and the distribution is very uniform, which proves that the device can achieve the goal of fast antibody entering the tissue.
Example 2:
staining of cleared brain tissue with Anti-Histone3 antibody
(a) Assembled sample cavity
According to the sample chamber from one end to the other end in proper order including: the sample cavity is assembled by the top cover, the first dialysis membrane, the inner ring, the bottom cover and the second dialysis membrane in sequence, wherein the inner ring comprises an upper agarose hollow cylinder, a sample and a lower agarose hollow cylinder, and the first dialysis membrane and the second dialysis membrane need to remove protective glycerol attached to the surfaces by deionized water.
Wherein the sample is a brain slice of adult mouse (C57BL/6) with thickness of 1mm and made transparent by passive Clarity method
The assembling of the sample chamber comprises the steps of:
(a-1) adding an antibody to a hollow portion of a lower agarose hollow cylinder;
(a-2) placing the sample in a hollow portion of a lower agarose hollow cylinder and injecting 8% agarose around the sample;
(a-3) the upper agarose hollow cylinder was mounted after agarose solidification, and the hollow part of the cylinder was filled with an antibody solution.
(b) Dyeing, wherein the dyeing comprises the following steps:
(b-1) preparing a 3% BSA blocking solution using the electrophoresis solution, and then performing the following steps: preparing a Histone3 antibody according to the proportion of 200;
(b-2) after the sample is installed, putting the sample cavity into an electrophoresis cavity, fixing the sample cavity by using a sample cavity fixing frame, adding a buffer solution, and covering the sample cavity;
(b-3) turning on a power supply, setting the current to be 25mA, and running for 1h for electrophoresis; then, exchanging the current direction, setting the current to be 25mA, and running for 1h for electrophoresis;
and (b-4) cutting off the power supply, and finishing the dyeing after incubating for 30 min.
(c) Antibody elution, wherein antibody elution comprises the steps of
(c-1) opening the top cover and the bottom cover, and using new buffer solution, wherein the buffer solution is sodium borate buffer solution, and the concentration of the buffer solution is 0.1mol/L, pH and is 8.5;
(c-2) excess antibody molecules that failed to bind effectively to the antigen (i.e., Histone3 protein) were removed by elution at 25mA for 30 min.
(d) Fluorescence imaging detection of marking effects
After the elution was completed, the sample was taken out from the inner ring, excess agarose was cut off, and the sample was peeled off and imaged by a laser scanning confocal microscope (A1Si, Nikon) at a magnification of 10 ×.
Imaging results as shown in figure 7, we imaged samples of this 1mm experimental group at intervals in order to reduce quenching of the antibody during imaging. As can be seen from the A and B panels, the antibodies of the experimental group all entered the sample and were distributed more uniformly. As can be seen from panel C, the electrophoretic removal of excess antibody works very well, and the signal of the sample's subsequent structural part is clear, with a clean background. As can be seen from the D, E and F plots, the antibody only entered a very thin layer inside the tissue after the control group had been free-diffusion stained for the same time, and it was difficult to stain the entire sample. This demonstrates that the rapid staining apparatus is very effective for thick tissues.
A: z-axis distribution of three-dimensional images of experimental group samples on a 1mm scale; b: the distribution of the xy direction of the three-dimensional images of the experimental group samples on the scale of 1 mm; c: microscopic images of experimental group samples at different depths; d: microscopic images of control samples at different depths; e: z-axis distribution of three-dimensional images of control samples on a 1mm scale; f: distribution of the control group samples in xy direction of the three-dimensional image on a 1mm scale.
Example 3
Rapid immunostaining of astrocytes in mouse brain tissue (including primary and secondary antibody staining)
(a) Assembling the sample chamber
According to the sample chamber from one end to the other end in proper order including: the sample cavity is assembled by the top cover, the first dialysis membrane, the inner ring, the bottom cover and the second dialysis membrane in sequence, wherein the inner ring comprises an upper agarose hollow cylinder, a sample and a lower agarose hollow cylinder, and the first dialysis membrane and the second dialysis membrane need to be removed with deionized water to protect glycerol attached to the surfaces.
Wherein the sample is a brain slice of adult mouse (C57BL/6) with thickness of 1mm
The assembling of the sample chamber comprises the steps of:
(a-1) adding an antibody to a hollow portion of a lower agarose hollow cylinder;
(a-2) placing the sample in a hollow portion of a lower agarose hollow cylinder and injecting 8% agarose around the sample;
(a-3) the agarose is solidified, then the upper agarose hollow cylinder is mounted, and the hollow portion is filled with the antibody solution.
(b) Primary and secondary antibody staining comprising the steps of:
(b-1) according to 1: preparing a GFAP primary antibody solution and a GFAP secondary antibody solution by using a BSA solution at a ratio of 200;
(b-2) GFAP primary staining was performed in the same manner as in example 2;
(b-3) after the primary anti-dyeing is completed, opening the top cover and the bottom cover of the mold, and sucking out a primary anti-solution;
(b-4) putting the inner ring and the sample which is arranged in the inner ring by the agarose into an electrophoresis tank, fixing the sample by the blue-butyl adhesive, and adding new electrophoresis solution;
(b-5) setting the current to be 25mA, running for 40min for electrophoresis, and taking out the inner ring;
(b-6) after absorbing the excess solution in the inner ring, adding a secondary antibody solution into the hollow parts of the upper and lower agarose cylinders respectively;
(b-7) after the first dialysis membrane is placed, tightly covering the first dialysis membrane with a top cover; finally, the whole sample cavity is tightly covered and leak detection is carried out;
(b-8) dyeing according to the electrophoresis condition of the primary anti-dyeing;
(b-9) opening the top cap and the bottom cap, and sucking out the residual secondary antibody solution;
(b-10) the mixture was eluted at 25mA for 45min to remove excess secondary antibody. Wherein the electrophoretic fluid is replaced each time the staining and the elution are alternated. After the end, the sample was removed from the inner ring, excess agarose was cut off, and the sample was peeled off and imaged with a laser scanning confocal microscope (A1Si, Nikon) at a magnification of 10X.
Imaging results as shown in fig. 8, we imaged samples of this 1mm experimental group at intervals in order to reduce quenching of the antibody during imaging. As can be seen from the A and B panels, the antibodies of the experimental group all entered the sample and were distributed more uniformly. As can be seen from panel C, the electrophoretic removal of excess antibody works very well, and the signal of the sample's subsequent structural part is clear, with a clean background. As can be seen from the D, E and F panels, the antibody only entered the surface when the control group was free-diffusion stained for the same time, and it was difficult to stain the entire sample. This demonstrates that the rapid staining apparatus is very effective for thick tissues.
A: z-axis distribution of three-dimensional images of experimental group samples on a 1mm scale; b: the distribution of the xy direction of the three-dimensional images of the experimental group samples on the scale of 1 mm; c: microscopic images of experimental group samples at different depths; d: microscopic images of control samples at different depths; e: z-axis distribution of three-dimensional images of control samples on a 1mm scale; f: distribution of the control group samples in xy direction of the three-dimensional image on a 1mm scale.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A device for realizing the quick staining of biological tissues is characterized by comprising a staining box;
wherein, the dyeing box is internally provided with 1 to 100 electrophoresis chamber placing grooves;
the electrophoresis cavity holding groove is provided with an electrophoresis cavity, a temperature sensor, an ion concentration sensor, a pH sensor, a current sensor and a flow velocity sensor;
the outer side of the notch of the electrophoresis cavity holding groove is provided with a positive electrode interface and a negative electrode interface;
the electrophoresis chamber is provided with a positive electrode, a negative electrode, a sample chamber fixing frame and a sample chamber; the device is provided with a control system, and the control system comprises a control panel, a temperature control module, a hydraulic control module, a current module, a power switch, a time control module and a pH control module;
the temperature control module, the hydraulic control module and the current module are positioned below an electrophoresis cavity placing groove in the device;
the temperature control module is connected with the temperature sensor;
the hydraulic control module is connected with the flow velocity sensor, the ion concentration sensor and the pH sensor;
the current module is connected with the power switch and the current sensor; and is
The power supply can provide the current intensity of 0.1-300mA, and the direction of the current can be changed alternately.
2. The device of claim 1, wherein the positive electrode is 0.01-0.5cm from the chamber wall and the negative electrode is 0.01-0.5cm from the chamber wall.
3. The device of claim 1, wherein the negative electrode comprises an electrode material and a fixed electrode material;
the fixed electrode material is an insulating material, and is selected from the following group: a glass plate, a plastic plate, a ceramic plate, or a combination thereof;
the electrode material is a conductive material, and is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof.
4. The apparatus of claim 1, wherein the positive electrode comprises an electrode material and a fixed electrode material:
the fixed electrode material is an insulating material, and is selected from the following group: a glass plate, a plastic plate, a ceramic plate, or a combination thereof;
the electrode material is a conductive material, and is selected from the following group: platinum, gold, silver, conductive glass, carbon, graphene, or combinations thereof.
5. The apparatus of claim 2, wherein the control panel comprises a temperature adjustment panel, an ion concentration adjustment panel, a pH adjustment panel, a current adjustment panel, a time adjustment panel, and a flow rate control panel.
6. The apparatus of claim 1, wherein the sample chamber comprises: the top cover, the first dialysis membrane, the inner ring, the bottom cover and the second dialysis membrane;
wherein, the inner ring inside include: an upper agarose hollow column, sample, lower agarose hollow column;
wherein, the sample chamber include from one end to the other end in proper order: top cap, first dialysis membrane, inner ring, bottom, second dialysis membrane.
7. The device of claim 6, wherein the first dialysis membrane has a molecular weight cut-off of 6-500kDa and the second dialysis membrane has a molecular weight cut-off of 6-500 kDa.
8. A method of using the apparatus of claim 1, the method comprising the steps of:
(I) placing an electrophoresis cavity containing buffer solution in an electrophoresis cavity placing groove, placing a sample cavity in a sample cavity fixing frame, and adjusting the current between a positive electrode and a negative electrode; wherein the buffer solution has an ion concentration of 0.1-10M, pH of 1-13 and a current intensity of 0.1-300 mA;
(II) dyeing for 30min-48 h.
9. The method of claim 8, wherein said method further comprises the steps of: eluting, wherein in the elution step, the ion concentration of the buffer solution is 0.1-10M, pH and is 1-13, the current intensity is 0-500mA, and the elution time is 30min-24 h.
10. The method of claim 8, wherein the current ranges from 0.1 mA to 300mA and the direction of the current is alternated.
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| CN201810368691.9A CN108572099B (en) | 2018-04-23 | 2018-04-23 | Method and device for realizing rapid staining of biological tissues |
| PCT/CN2019/083950 WO2019206150A1 (en) | 2018-04-23 | 2019-04-23 | Method and device capable of realizing rapid staining of biological tissue |
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| 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 |
| CN113049804B (en) * | 2021-03-17 | 2022-09-09 | 上海交通大学 | Method for rapidly marking biological tissues |
| KR102646529B1 (en) | 2021-10-08 | 2024-03-13 | 서울대학교병원 | Kit for staining tissue for on-site evaluation |
| CN114252324B (en) * | 2022-03-02 | 2022-05-13 | 中国人民解放军军事科学院军事医学研究院 | Device is hatched to porous brain piece that facilitates use |
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| US9395330B2 (en) * | 2007-03-19 | 2016-07-19 | Case Western Reserve University | Degradable polyacrylamide gel |
| CN101970111B (en) * | 2007-06-21 | 2013-09-11 | 简·探针公司 | Instrument and receptacles for performing processes |
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