CN108051582A - A kind of immunohistochemistry of improvement repairs liquid - Google Patents
A kind of immunohistochemistry of improvement repairs liquid Download PDFInfo
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- CN108051582A CN108051582A CN201711286273.7A CN201711286273A CN108051582A CN 108051582 A CN108051582 A CN 108051582A CN 201711286273 A CN201711286273 A CN 201711286273A CN 108051582 A CN108051582 A CN 108051582A
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Abstract
A kind of immunohistochemistry the present invention relates to improvement repairs liquid, including：Trisodium citrate, NP 40 and emulsifier.The immunohistochemistry of the improvement of the present invention repairs liquid infiltration, and repairing effect is preferable, histotomy can be repaired under less dosage；The immunohistochemistry of the improvement of the present invention repairs the antigen that liquid is suitable for many clinical more difficult detections, such as Fas, Bax, FVIII, Lamnin, CoIV, Keratin, CEA, GFAP etc.；Histotomy easily dyes after the immunohistochemistry of improvement using the present invention repairs liquid reparation, and coloring is preferable.
Description
Technical field
The present invention relates to field of immunology more particularly to a kind of immunohistochemistry of improvement to repair liquid.
Background technology
Antigen is to refer to that body is stimulated to generate (specificity) immune response, and can be with immune response product antibodies and cause
Quick lymphocyte combines in vivo and in vitro, and the substance of immunological effect (specific reaction) occurs.The fundamental property of antigen has foreign matter
Property, macromoleculariness and specificity.The foreign body antigenic substance referred into body tissue, it is necessary to the body tissue cell
Ingredient differs.Antigen is generally referred into the in vivo foreign substance of machine, such as bacterium, virus, pollen；Antigen can also be not
The substance of jljl inter-species, if the serum of horse enters the internal of rabbit, many protein in horse serum just become the antigen of rabbit
Substance；Substance between allogeneic can also become antigen, such as blood group, trnasplantion immunity；From in vivo some isolation ingredients
Antigen, such as eyes crystalline protein, spermatid, thyroglobulin can be become, under normal circumstances, be integrally fixed at machine
The a certain position of body, the cell with generating antibody are isolated, therefore will not cause self generation antibody.Immunohistochemistry is clinical disease
The most commonly used method in reason diagnosis, principle is to be combined using antigen with antibody specificity, resists mark by chemical reaction
The chromogenic reagent of body determines histocyte endoantigen (peptide and protein), it is positioned, qualitative and quantitative is ground
Study carefully.Immunohistochemical experiment predominantly two major class of tissue specimen and cell specimen used, paraffin section are that making tissue specimen is most normal
With, most basic prefered method, paraffin section manufacturing process has a certain impact to tissue endoantigen exposure, but can carry out antigen
It repairs.
Paraffin section sample is fixed with formaldehyde so that intracellular antigen formed aldehyde key, carboxylic first key and be closed part
Antigenic determinant, while crosslinked between albumen and make antigenic determinant hidden.So when carrying out immunohistochemical staining, elder generation is needed
Carry out antigen retrieval.Antigen retrieval is most important in immunohistochemical staining, directly determines coloration result and diagnostic result.
Current immunohistochemistry is repaired there are many kinds of liquid, as the immunohistochemistry of DAKO DENMARK A/S companies repair liquid,
Shanghai Long Island antibody diagnosing reagent Co., Ltd, Shanghai Chang-dao Bioengineering Co., Ltd., Foochow steps neoformation technological development has
Limit company produces citrate immunohistochemistry and repairs liquid etc..Immunohistochemistry repairs liquid ingredient：Citric acid, trisodium citrate are prevented
Rotten agent etc..
Above-mentioned immunohistochemistry repairs liquid based on citric acid and the common primary solvent of clinical immunization group, but only suitable
For general antigen retrieval.The more difficult detection of clinical many antigens, the repairing effect that conventional immunohistochemistry repairs liquid is bad, directly
Connect the follow-up dyeing of influence and diagnosis.
The content of the invention
To solve the above problems, the immunohistochemistry the present invention provides a kind of improvement repairs liquid.The improvement of the present invention is exempted from
Epidemic disease groupization reparation liquid repairing effect is preferable, can play good repair to the antigen of much more difficult detections.
According to an aspect of the present invention, a kind of immunohistochemistry of improvement provided by the invention repairs liquid, including：Citric acid
Trisodium, NP-40 and emulsifier.
Further, the component of following parts by weight is included：20-40 parts of trisodium citrates, 0.1-4 parts of NP-40,2-20 parts of breasts
Agent.
Further, the component of following parts by weight is included：25-35 parts of trisodium citrates, 0.5-2 parts of NP-40,5-15 parts of breasts
Agent.
Further, the component of following parts by weight is included：30 parts of trisodium citrates, 0.75 part of NP-40,7.5 parts of emulsifiers.
Further, the emulsifier is Tween-20 or glycerine.
Further, the preparation method of the immunohistochemistry reparation liquid comprises the following steps：Using H20 dissolving citric acid three
Sodium adds in NP-40；Until completely dissolved, emulsifier is added in.
Further, the emulsifier is Tween-20 or glycerine.
Compared with prior art, the present invention it has the following advantages：
1. the immunohistochemistry of the improvement of the present invention repairs liquid infiltration, repairing effect is preferable, can be right under less dosage
Histotomy is repaired；
2. the immunohistochemistry of the improvement of the present invention repairs the antigen that liquid is suitable for many clinical more difficult detections, such as Fas,
Bax, FVIII, Lamnin, CoIV, Keratin, CEA, GFAP etc.；
3. histotomy easily dyes after the immunohistochemistry of improvement using the present invention repairs liquid reparation, coloring compared with
It is good；
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, and in order to allow above and other objects of the present invention, feature and advantage can
It is clearer and more comprehensible, below the special specific embodiment for lifting the present invention.
Specific embodiment
Technical scheme is clearly and completely described below in conjunction with embodiment, it is clear that described reality
It is part of the embodiment of the present invention to apply example, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel all other embodiments obtained without making creative work belong to the model that the present invention protects
It encloses.
A kind of immunohistochemistry of the improvement provided in the present embodiment repairs liquid, including：Trisodium citrate, NP-40 and emulsification
Agent.The present invention improvement immunohistochemistry repair liquid using trisodium citrate as main repairing activity ingredient, and with NP-40,
Tween-20 is with H2O as mixed solvent.
As described above, emulsifier plays emulsification, the compatibility for repairing each substance in liquid can be improved, and to repairing liquid
In active ingredient influence it is smaller, the reparation of histotomy will not be caused excessively to influence.Wherein, the emulsifier is to spit
Temperature -20 or glycerine.
Polysorbas20 is a kind of surfactant, it is a kind of macromolecular, existing hydrophilic part on molecule, and has oleophylic
Part.So plant absorption undissolvable macromolecular in water can be promoted, it can also help moisture through some containing the high life of fat
Object film.
Glycerine, i.e. glycerine, the pleasantly sweet thick liquid of no color or smell have hypertonicity, can be miscible in any proportion with water,
There is great hygroscopicity, for quickly removing intracellular water, play the role of protecting antigen.
NP-40 (Nonidet P 40), Chinese is translated into Nonidet P40, is a kind of mild non-ionic de-sludging
Agent；NP-40 is a kind of very mild Nonionic Detergents, and 1% concentration can destroy cell membrane substantially, and nuclear membrane is broken
Bad effect is weaker, and plasmosin can be obtained with reference to specific buffer solution.It is strong with protein binding power, for preventing material molecule from dredging
It interacts between water, it is ensured that the abundant dissolving of albumen and stable structure.Particularly for memebrane protein it is non denatured under the conditions of dissolving.
NP-4 plays the role of display to intracellular antigen, can improve the degree of exposure of more difficult detection antigen.
The immunohistochemistry of the improvement of the present invention repairs the H in liquid2O can be distilled water, or tri-distilled water；Distilled water
It is one kind of redistilled water, is by the water after single flash, distills obtained water again；Tri-distilled water is by distilling three times
Water.
Embodiment 1
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：30g trisodium citrates, 0.75mLNP-40,7.5mL are spat
The distilled water of temperature -20 and surplus.
Preparation method is as follows：Trisodium citrate is weighed, after being dissolved with distilled water；NP-40 is added in, is added again after being completely dissolved
Tween-20 is settled to rated capacity after Tween-20 dissolving with distilled water.
Embodiment 2
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：20g trisodium citrates, 4mL NP-40,2mL glycerine and
The distilled water of surplus.
Preparation method is as follows：Trisodium citrate is weighed, after being dissolved with tri-distilled water；NP-40 is added in, is added again after being completely dissolved
Glycerine is settled to rated capacity after glycerine dissolving with distilled water.
Embodiment 3
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：40g trisodium citrates, 0.1mL NP-40,20mL are spat
The distilled water of temperature -20 and surplus.
Preparation method is the same as embodiment 1
Embodiment 4
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：25g trisodium citrates, 2mL NP-40,5mL glycerine and
The distilled water of surplus.
Preparation method is the same as embodiment 2
Embodiment 5
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：35g trisodium citrates, 0.5mL NP-40,15mL are spat
The distilled water of temperature -20 and surplus.
Preparation method is the same as embodiment 1
Embodiment 6
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：23g trisodium citrates, 3mL NP-40,4mL Tween-20s
With the distilled water of surplus.
Preparation method is the same as embodiment 1
Embodiment 7
Per 1000mL, the immunohistochemistry of improvement is repaired liquid and is included：38g trisodium citrates, 0.2mL NP-40,17mL glycerine
With the distilled water of surplus.
Preparation method is the same as embodiment 2
The antigen retrieval of histotomy of the test example 1 containing GEA antigens
Test A：Liquid is repaired using the immunohistochemistry of improvement of the present invention
1) histotomy is put into dimethylbenzene and dewaxed 5 minutes；Section is sequentially placed into 100% volume ratio alcohol after dewaxing
2 minutes, 90% volume ratio alcohol 2 minutes, 80% volume ratio alcohol 2 minutes and 70% volume ratio alcohol 2 minutes；Buffer
(0.025mol/L trisodium citrates) rinses 5 minutes；
2) immunohistochemistry of the improvement of embodiment 1 is repaired into liquid and is heated at high temperature to 92 DEG C, section is placed and is repaired in liquid, lid
Upper cover starts timing, and after 2 minutes natural cooling 30 minutes at room temperature；Buffer is rinsed 5 minutes；
3) peroxidase blocking reagent 5 minutes；Buffer is rinsed 5 minutes；
4) primary antibody (commercially available mouse anti-human monoclonal's antibody) is incubated 20 minutes；Buffer is rinsed 5 minutes；
5) secondary antibody (commercially available enzyme mark sheep anti-mouse igg polymer) is incubated 20 minutes；Buffer is rinsed 5 minutes；
6) through DAB 10 points of kinds of colour developing；Tap water rinses；
7) redyed 5 minutes through haematoxylin；Tap water rinses；
8) section is sequentially placed into 70% volume ratio alcohol 2 minutes, 80% volume ratio alcohol 2 minutes, 90% volume ratio wine
Essence 2 minutes, each 2 minutes of 100% volume ratio alcohol；Transparent 5 minutes of dimethylbenzene, mounting.
Test B：Use the antigen retrieval buffers described in other non-invention
Antigen retrieval buffers are selected from：Fuzhou Maixin biotechnology Development Co., Ltd produces citrate antigen retrieval buffers.
Test procedure：
A) paraffin section is placed in fresh dimethylbenzene, is impregnated 15 minutes × 2 times；After removing extra liquid, put anhydrous
In ethyl alcohol, impregnate 3 minutes × 2 times；It after removing extra liquid, puts in 95% ethyl alcohol, impregnates 3 minutes；Remove extra liquid
Afterwards, put in 85% ethyl alcohol, impregnate 3 minutes；Tap water rinses 1 minute；PBS solution is rinsed 3 minutes × 3 times.
B) opening cover of pressure-cooker high-power heating citric acid tissue antigen recovery liquid on electromagnetic oven extremely seethes with excitement；To dewax aquation
Section afterwards is placed on high temperature resistant staining rack, is put into pressure cooker；Pot cover is covered, buckles pressure valve, continues to be heated to spray vapour, open
After beginning timing 2 minutes, pressure cooker leaves heat source, natural cooling 5 minutes；It is allowed to cool down with tap water shower, valve is gone to uncap, treat pot
Section is taken out after middle liquid cooled to room temperature；Distilled water flushing 3 minutes × 2 times；It is rinsed 3 minutes × 3 times with PBS solution.
C) PBS solution is removed, in the test serum region of oil pike delineation plus 100 μ l endogenous peroxydases block
Agent is incubated 10 minutes at room temperature.PBS solution is rinsed 3 minutes × 3 times.
D) PBS solution is removed, adds the primary antibody of 100 μ l or blank control reagent, is incubated 60 minutes at room temperature.PBS solution is rushed
It washes 3 minutes × 3 times.
E) PBS solution is removed, adds 100 μ l enzyme mark sheep anti-mouse igg polymer, is incubated 15 minutes at room temperature.PBS solution is rinsed
3 minutes × 3 times.
F) PBS solution is removed, adds the DAB developing solutions of 100~200 μ l Fresh, is incubated 3~5 minutes, om observation dye
Color result is usually no more than 10 minutes.
G) tap water rinses, and 100~200 μ l haematoxylin body cells dyeing liquors is added to be incubated 10~30 seconds.PBS solution or originally
Indigo plant is returned in water flushing.
The experiment conclusion of test example 1：Above two method is compared, and the section that experiment A is repaired, GEA is positive, and positioning is clear and definite, the back of the body
Scape is clean, dark-brown is shown after dyeing, good contrast, there are few flakes；The section that B is repaired is tested, the GEA positives are basically completed positioning,
But light brown is shown after dyeing, flake rate is higher, so in the antigen retrieval contrast experiment of the histotomy containing GEA antigens,
The immunohistochemistry of improvement of the present invention repairs effect of the effect better than the antigen retrieval buffers described in experiment B of liquid.
Histotomy antigen retrieval of the test example 2 containing GFAP antigens
Test C：Liquid is repaired using the immunohistochemistry of improvement of the present invention
1) histotomy is put into dimethylbenzene to dewax, 5 minutes；100% alcohol, 90% alcohol, 80% alcohol, 70% alcohol
Each 2 minutes of gradient；Buffer is rinsed 5 minutes；
2) immunohistochemistry of improvement of the present invention is first repaired into liquid and is heated at high temperature to 92 DEG C, section is placed and repairs liquid
Interior, the heating that closes the lid, which is heated to steaming, starts timing 2 minutes, natural cooling 30 minutes；Buffer is rinsed 5 minutes；
3) peroxidase blocking reagent 5 minutes；Buffer is rinsed 5 minutes；
4) primary antibody (commercially available mouse anti-human monoclonal's antibody) is incubated 20 minutes；Buffer is rinsed 5 minutes；
5) secondary antibody (commercially available enzyme mark sheep anti-mouse igg polymer) is incubated 20 minutes；Buffer is rinsed 5 minutes；
6) 10 points of kinds of DAB colour developings；Tap water rinses；
7) haematoxylin is redyed 5 minutes；Tap water rinses；
8) section is sequentially placed into 70% alcohol, 80% alcohol, 90% alcohol, each 2 minutes of 100% alcohol gradient；Diformazan
Transparent 5 minutes of benzene, mounting.
Test D：Use the antigen retrieval buffers described in other non-invention
Antigen retrieval buffers are selected from：Fuzhou Maixin biotechnology Development Co., Ltd produces citrate antigen retrieval buffers.
Test procedure：
A) paraffin section is placed in fresh dimethylbenzene, is impregnated 15 minutes × 2 times；After removing extra liquid, put anhydrous
In ethyl alcohol, impregnate 3 minutes × 2 times；It after removing extra liquid, puts in 95% ethyl alcohol, impregnates 3 minutes；Remove extra liquid
Afterwards, put in 85% ethyl alcohol, impregnate 3 minutes；Tap water rinses 1 minute；PBS solution is rinsed 3 minutes × 3 times.
B) opening cover of pressure-cooker high-power heating citric acid tissue antigen recovery liquid on electromagnetic oven extremely seethes with excitement；To dewax aquation
Section afterwards is placed on high temperature resistant staining rack, is put into pressure cooker；Pot cover is covered, buckles pressure valve, continues to be heated to spray vapour, open
After beginning timing 2 minutes, pressure cooker leaves heat source, natural cooling 5 minutes；It is allowed to cool down with tap water shower, valve is gone to uncap, treat pot
Section is taken out after middle liquid cooled to room temperature；Distilled water flushing 3 minutes × 2 times；It is rinsed 3 minutes × 3 times with PBS solution.
C) PBS solution is removed, in the test serum region of oil pike delineation plus 100 μ l endogenous peroxydases block
Agent is incubated 10 minutes at room temperature.PBS solution is rinsed 3 minutes × 3 times.
D) PBS solution is removed, adds the primary antibody of 100 μ l or blank control reagent, is incubated 60 minutes at room temperature.PBS solution is rushed
It washes 3 minutes × 3 times.
E) PBS solution is removed, adds 100 μ l enzyme mark sheep anti-mouse igg polymer, is incubated 15 minutes at room temperature.PBS solution is rinsed
3 minutes × 3 times.
F) PBS solution is removed, adds the DAB developing solutions of 100~200 μ l Fresh, is incubated 3~5 minutes, om observation dye
Color result is usually no more than 10 minutes.
G) tap water rinses, and 100~200 μ l haematoxylin body cells dyeing liquors is added to be incubated 10~30 seconds.PBS solution or originally
Indigo plant is returned in water flushing.
The experiment conclusion of test example 2：Above two method is compared, and the section that experiment A is repaired, GFAP is positive, and positioning is clear and definite,
Clean background shows dark-brown after dyeing, good contrast, there are few flakes；The section that B is repaired is tested, GFAP is positive, and positioning is unclear,
Non-specificity coloring is more, relatively fuzzyyer after dyeing, slightly flake, so in the histotomy antigen retrieval containing GFAP antigens
Contrast experiment in, the immunohistochemistry of improvement of the present invention repairs antigen retrieval buffers of the effect of liquid described in better than experiment D
Effect.
The immunohistochemistry that 3 present invention of test example improves repairs the composition selection of liquid
The additive amount of each component in 1 test example of table
Experiment 3-1 is weighed to 3-5 is tested according to each component content described in form, and according to 1 the method for embodiment
Be prepared into 5 kinds improvement immunohistochemistry repair liquid, by 5 kinds improvement immunohistochemistry repair liquid according to test example 1 test A methods into
Row experiment.
The experimental result of experiment 3：Experiment 3-1 realizes preferable positive positioning to 3-5 is tested, and is shown after experiment 3-1 dyeing
Light brown has flake；It shows light brown after testing 3-2 dyeing, there is flake；Dark-brown, clear background, nothing are shown after testing 3-3 dyeing
Flake；Dark-brown, slightly clear background, flake are shown after testing 3-4 dyeing；Light brown is shown after testing 3-5 dyeing, background occasionally has dry
Disturb color, no flake；So in the contrast experiment of the histotomy antigen retrieval containing GFAP antigens, improvement described in 3-3 is tested
Immunohistochemistry repair liquid effect better than other group improvement immunohistochemistry repair liquid effect.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution recorded in foregoing embodiments either to which part or all technical characteristic into
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is not made to depart from various embodiments of the present invention technology
The scope of scheme.
Claims (7)
1. a kind of immunohistochemistry of improvement repairs liquid, it is characterised in that：Including：Trisodium citrate, NP-40 and emulsifier.
2. the immunohistochemistry improved according to claim 1 repairs liquid, which is characterized in that includes the component of following parts by weight：
20-40 parts of trisodium citrates, 0.1-4 parts of NP-40,2-20 parts of emulsifiers.
3. the immunohistochemistry improved according to claim 2 repairs liquid, which is characterized in that includes the component of following parts by weight：
25-35 parts of trisodium citrates, 0.5-2 parts of NP-40,5-15 parts of emulsifiers.
4. the immunohistochemistry improved according to claim 3 repairs liquid, which is characterized in that includes the component of following parts by weight：
30 parts of trisodium citrates, 0.75 part of NP-40,7.5 parts of emulsifiers.
5. the immunohistochemistry that improves according to claim 1 repairs liquid, which is characterized in that the emulsifier for Tween-20 or
Glycerine.
6. the immunohistochemistry improved according to claim 1 repairs liquid, which is characterized in that the immunohistochemistry repairs the system of liquid
Preparation Method includes step：
Using H2O dissolves trisodium citrate, adds in NP-40；Until completely dissolved, emulsifier is added in.
7. the immunohistochemistry that improves according to claim 6 repairs liquid, which is characterized in that the emulsifier for Tween-20 or
Glycerine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346552A (en) * | 2019-07-25 | 2019-10-18 | 深圳褀氏生物医疗电子有限公司 | A kind of universal antigen retrieval buffer |
| CN113495138A (en) * | 2021-06-22 | 2021-10-12 | 南京弗瑞思生物科技有限公司 | Tissue slice antigen repair liquid and use method thereof |
| CN113686634A (en) * | 2021-08-10 | 2021-11-23 | 杭州迪英加科技有限公司 | Dewaxing repair liquid beneficial to acquisition of paraffin section images |
| CN113740522A (en) * | 2021-09-01 | 2021-12-03 | 陕西脉元生物科技有限公司 | Antigen repairing liquid, cell climbing sheet prepared by using antigen repairing liquid, and preparation method and application of cell climbing sheet |
| CN114295826A (en) * | 2021-12-22 | 2022-04-08 | 上海思路迪医学检验所有限公司 | Exosome cracking reagent and exosome PD-L1 protein detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007841A2 (en) * | 2004-07-23 | 2006-01-26 | Dakocytomation Denmark A/S | Heat-induced antigen retrieval methods and compositions therefor |
| CN105987839A (en) * | 2016-06-03 | 2016-10-05 | 浙江世纪康大医疗科技股份有限公司 | EDTA-citric acid antigen repair fluid |
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2017
- 2017-12-07 CN CN201711286273.7A patent/CN108051582B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007841A2 (en) * | 2004-07-23 | 2006-01-26 | Dakocytomation Denmark A/S | Heat-induced antigen retrieval methods and compositions therefor |
| CN105987839A (en) * | 2016-06-03 | 2016-10-05 | 浙江世纪康大医疗科技股份有限公司 | EDTA-citric acid antigen repair fluid |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346552A (en) * | 2019-07-25 | 2019-10-18 | 深圳褀氏生物医疗电子有限公司 | A kind of universal antigen retrieval buffer |
| CN113495138A (en) * | 2021-06-22 | 2021-10-12 | 南京弗瑞思生物科技有限公司 | Tissue slice antigen repair liquid and use method thereof |
| CN113686634A (en) * | 2021-08-10 | 2021-11-23 | 杭州迪英加科技有限公司 | Dewaxing repair liquid beneficial to acquisition of paraffin section images |
| CN113740522A (en) * | 2021-09-01 | 2021-12-03 | 陕西脉元生物科技有限公司 | Antigen repairing liquid, cell climbing sheet prepared by using antigen repairing liquid, and preparation method and application of cell climbing sheet |
| CN114295826A (en) * | 2021-12-22 | 2022-04-08 | 上海思路迪医学检验所有限公司 | Exosome cracking reagent and exosome PD-L1 protein detection kit |
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| Publication number | Publication date |
|---|---|
| CN108051582B (en) | 2020-08-04 |
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