CN110257483B - Hybridization solution for in situ hybridization, preparation method thereof and detection kit - Google Patents
Hybridization solution for in situ hybridization, preparation method thereof and detection kit Download PDFInfo
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- CN110257483B CN110257483B CN201910617971.3A CN201910617971A CN110257483B CN 110257483 B CN110257483 B CN 110257483B CN 201910617971 A CN201910617971 A CN 201910617971A CN 110257483 B CN110257483 B CN 110257483B
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- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
Abstract
The invention relates to a hybridization solution for in situ hybridization, a preparation method thereof and an in situ hybridization detection kit. The hybridization solution for in situ hybridization comprises the following components in final concentration: formamide with the volume percentage concentration of not more than 2 percent, fluoride with the weight volume percentage concentration of 0.1-10 percent, SSC with the concentration of 2-4 times, EDTA with the concentration of 1-10mM, Denhardts solution with the concentration of 1-5 times, dextran sulfate with the weight volume percentage concentration of 5-10 percent, salmon sperm DNA with the concentration of 0.1-1mg/mL, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution or Tris-HCl buffer solution with the concentration of 50-100mM and the pH value of 7.0. The hybridization solution for in-situ hybridization can improve the hybridization efficiency and shorten the hybridization time under the condition of ensuring a hybridization signal.
Description
Technical Field
The invention relates to an in-situ hybridization technology, in particular to a hybridization solution for in-situ hybridization, a preparation method thereof and an in-situ hybridization detection kit.
Background
In 1969 Gall and Pardie used isotope-labeled probes to determine nucleic acid sequences In intact cells for the first time, and In Situ Hybridization (ISH) was rapidly developed. The advent of immunohistochemical staining procedures has facilitated the development of non-isotopically labeled nucleic acid probes, enabling the detection of DNA and RNA in tissues that were conventionally formalin-fixed and paraffin-embedded. In formalin-fixed tissues, formaldehyde penetrates between nucleic acids and proteins, which can lead to DNA-DNA, DNA-protein cross-linking, preserving the antigen in situ. In situ hybridization is a technique combining molecular hybridization and histology, wherein a labeled probe such as biotin, digoxigenin and the like is adopted, and under a proper condition, a DNA-DNA, DNA-RNA or RNA-RNA hybrid double chain is formed through hydrogen bonding, a signal amplification system is further adopted to amplify the hybridized probe, a color development system develops color, and finally the hybridized signal is observed under a microscope. The signal amplification system and the chromogenic system are identical to the immunohistochemical antigen-antibody reaction. In situ hybridization includes Chromogenic In Situ Hybridization (CISH) and Fluorescent In Situ Hybridization (FISH). Chromogenic in situ hybridization uses non-fluorescent labels to label the probes, such as digoxigenin, biotin, etc., and therefore requires a relevant detection system to amplify the signal, which can be permanently preserved if the hybridization signal is developed with DAB. The fluorescent marker for fluorescence in situ hybridization is used for marking the probe, does not need a signal amplification process, and has the advantages of high sensitivity and simultaneous use of a plurality of probes. The in situ hybridization technique can detect the information of nucleic acid level in cells, such as infectious diseases, cytogenetic diseases and tumor-related chromosome abnormality, and can complement the application of immunohistochemistry, so that the in situ hybridization technique becomes a routine detection project of many hospital pathology departments.
The whole experimental operation process of chromogenic in situ hybridization comprises the following steps: dewaxing, hydrating, endogenous enzyme blocking, washing, protease digestion, washing, probe hybridization, washing, primary antibody incubation, washing, secondary antibody incubation, washing, color development, washing, lining dyeing, washing, dewatering, mounting and the like. The whole experimental operation process of fluorescence in situ hybridization comprises the following steps: dewaxing, hydrating, heat repairing, cleaning, protease digestion, cleaning, dehydrating, probe hybridization, cleaning, dehydrating, counterstaining and the like. The probe hybridization process is divided into denaturation and annealing. Denaturation refers to a process in which the hydrogen chains of complementary bases between double strands are dissociated into single strands when a double-stranded DNA molecule is heated to 92 ℃ C (90 ℃ C. -98 ℃ C.) under alkaline conditions or a denaturing agent is added. Annealing refers to the process of connecting the hydrogen bonds between complementary bases of a double-stranded nucleic acid molecule (probe) at a certain ion concentration and gradually reducing the temperature. During the annealing process, single-stranded DNA or RNA segments with exogenous and complementary sequences are added, and the single-stranded DNA or RNA segments can also be connected with one single-stranded complementary which is originally untwisted to form a heterogeneous double-stranded nucleic acid molecule, and the process is called nucleic acid hybridization. The effect of probe hybridization depends on signal intensity and hybridization background, which in turn is related to protease digestion, hybridization temperature, probe sequence, and hybridization solution for probe dilution.
The probe hybridization time of the in situ hybridization products on the market at present is mostly 4 to 16 hours, but the shorter the hybridization time, the less sufficient the hybridization, the weaker the hybridization signal intensity and the high nonspecific background, in this case, the rapid hybridization for 4 hours may reduce the sensitivity of the in situ hybridization, affecting the clinical differential diagnosis. Therefore, how to improve the hybridization efficiency, shorten the hybridization time, and improve the work efficiency of the pathology department under the condition of ensuring the hybridization signal intensity is particularly critical.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the hybridization solution for in-situ hybridization is provided, so that the hybridization efficiency can be improved and the hybridization time can be shortened under the condition of ensuring a hybridization signal;
furthermore, the invention also provides a preparation method of the hybridization solution for in situ hybridization; and an in situ hybridization detection kit based on the hybridization solution for in situ hybridization.
In order to solve the technical problems, the invention adopts the technical scheme that:
a hybridization solution for in situ hybridization, comprising the following components in final concentrations: formamide with the volume percentage concentration of not more than 2 percent, fluoride with the weight volume percentage concentration of 0.1-10 percent, SSC with the concentration of 2-4 times, EDTA with the concentration of 1-10mM, Denhardts solution with the concentration of 1-5 times, dextran sulfate with the weight volume percentage concentration of 5-10 percent, salmon sperm DNA with the concentration of 0.1-1mg/mL, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution or Tris-HCl buffer solution with the concentration of 50-100mM and the pH value of 7.0.
An in situ hybridization detection kit comprises an in situ hybridization detection probe, water and the hybridization solution for in situ hybridization.
The preparation method of the hybridization solution for in situ hybridization comprises the following steps:
(1) mixing 1.25g of sodium fluoride, 2.5mL of 1M disodium hydrogenphosphate-sodium dihydrogenphosphate buffer, 5mL of 20-fold SSC solution, 0.1mL of 0.5M EDTA solution, 1mL of 50-fold Denhardts solution and 2.5g of dextran sulfate, wherein the pH of the disodium hydrogenphosphate-sodium dihydrogenphosphate buffer is 7.0;
(2) carrying out centrifugal treatment on the mixed solution obtained in the step (1);
(3) salmon sperm DNA was added to a final concentration of 0.1mg/mL, followed by addition of ultrapure water to a volume of 50 mL.
The invention has the beneficial effects that:
(1) in the design of the components of the hybridization solution, the fluoride is adopted to replace or reduce the concentration of formamide (which can be reduced to below 2 percent or even 0 percent from the existing 20 percent), and the formamide is a toxic substance, so that the method can reduce or even avoid the use of formamide and has the advantage of environmental protection;
(2) the denaturation temperature can be reduced by 10-26 ℃, denaturation is carried out at a lower temperature, so that the tissue form can be better maintained, the stability of DNA can be improved, and the damage to the DNA can be reduced; meanwhile, because the concentration of the probe is usually far higher than that of the target DNA, the hybridization temperature is increased by 10-26 ℃ in the presence of fluoride ions, and the stable double strand formed by the probe and the nucleic acid is still not influenced; therefore, when the hybridization solution is applied to a hybridization process, the hybridization temperature can be increased, so that the hybridization speed can be increased, the hybridization specificity can be improved, the background can be reduced, the in-situ hybridization can be carried out more quickly, and the signal-to-background ratio is clearer;
(3) the hybridization solution can be suitable for hybridization of different probes including DNA, RNA and the like, and the hybridization time can be effectively shortened, the hybridization effect is optimized, and the background is reduced by using the formula of the hybridization solution.
Drawings
FIG. 1 is a diagram showing the results of Kappa in situ hybridization in a hybridization solution for in situ hybridization according to an embodiment of the present invention under a condition of hybridization at 50 ℃ for 90 minutes;
FIG. 2 is a diagram showing the results of Kappa in situ hybridization in the hybridization solution for in situ hybridization according to the embodiment of the present invention under the overnight hybridization condition at 37 ℃;
FIG. 3 is a graph showing the results of in situ hybridization of Lambda using a hybridization solution for in situ hybridization according to an embodiment of the present invention at 50 ℃ for 90 minutes;
FIG. 4 is a graph showing the results of in situ hybridization of Lambda in a hybridization solution for in situ hybridization according to an embodiment of the present invention under overnight hybridization conditions at 37 ℃;
FIG. 5 is a graph showing the results of HER2 fluorescence in situ hybridization performed by a hybridization solution for in situ hybridization at 66 ℃ for 2 hours according to an embodiment of the present invention;
FIG. 6 is a graph showing the results of fluorescent in situ hybridization of HER2 in a hybridization solution for in situ hybridization at 50 ℃ overnight according to an embodiment of the present invention.
Detailed Description
In order to explain the technical contents, the objects and the effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key concept of the invention is as follows: the hybridization solution component is added with fluoride to reduce or replace formamide component.
Referring to FIGS. 1-6, a hybridization solution for in situ hybridization, the hybridization solution comprising the following components in final concentrations: formamide with the volume percentage concentration of not more than 2 percent, fluoride with the weight volume percentage concentration of 0.1-10 percent, SSC with the concentration of 2-4 times, EDTA with the concentration of 1-10mM, Denhardts solution with the concentration of 1-5 times, dextran sulfate with the weight volume percentage concentration of 5-10 percent, salmon sperm DNA with the concentration of 0.1-1mg/mL, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution or Tris-HCl buffer solution with the concentration of 50-100mM and the pH value of 7.0.
An in situ hybridization detection kit comprises an in situ hybridization detection probe, water and the hybridization solution for in situ hybridization.
The preparation method of the hybridization solution for in situ hybridization comprises the following steps:
(1) mixing 1.25g of sodium fluoride, 2.5mL of 1M disodium hydrogenphosphate-sodium dihydrogenphosphate buffer, 5mL of 20-fold SSC solution, 0.1mL of 0.5M EDTA solution, 1mL of 50-fold Denhardts solution and 2.5g of dextran sulfate, wherein the pH of the disodium hydrogenphosphate-sodium dihydrogenphosphate buffer is 7.0;
(2) carrying out centrifugal treatment on the mixed solution obtained in the step (1);
(3) salmon sperm DNA was added to a final concentration of 0.1mg/mL, followed by addition of ultrapure water to a volume of 50 mL.
The main mechanism of action of the hybridization solution for in situ hybridization of the present invention will now be explained:
compared with the conventional hybridization solution, the hybridization solution for in situ hybridization increases the component of fluoride to reduce or replace the component of formamide.
Like oxygen, nitrogen, chlorine, etc., fluorine atoms can form stable hydrogen bonds with hydrogen atoms, which are the primary factors in maintaining the stability of the double helix structure of DNA. The atomic radius of fluorine is closest to the size of hydrogen atom, and can form a stronger hydrogen bond than any other atom (oxygen, nitrogen, chlorine, sulfur, phosphorus) and the like. The fluorine ions are added into the hybridization solution, so that the opened double strand of DNA can be effectively prevented, the original double strand structure is restored, the denatured DNA is kept in a double strand separation state, and higher probability is provided for probe interventional hybridization. Meanwhile, the fluorine ions also promote the dissociation of double strands of the DNA, so that the denaturation temperature of the DNA can be carried out at a lower temperature in the in-situ hybridization process, the stability of the DNA and the probe is facilitated, and the tissue morphology is kept.
From the above description, the beneficial effects of the present invention are:
(1) in the design of the components of the hybridization solution, the fluoride is adopted to replace or reduce the concentration of formamide (which can be reduced to below 2 percent or even 0 percent from the existing 20 percent), and the formamide is a toxic substance, so that the method can reduce or even avoid the use of formamide and has the advantage of environmental protection;
(2) the denaturation temperature can be reduced by 10-26 ℃, denaturation is carried out at a lower temperature, so that the tissue form can be better maintained, the stability of DNA can be improved, and the damage to the DNA can be reduced; meanwhile, because the concentration of the probe is usually far higher than that of the target DNA, the hybridization temperature is increased by 10-26 ℃ in the presence of fluoride ions, and the stable double strand formed by the probe and the nucleic acid is still not influenced; therefore, when the hybridization solution is applied to a hybridization process, the hybridization temperature can be increased, so that the hybridization speed can be increased, the hybridization specificity can be improved, the background can be reduced, the in-situ hybridization can be carried out more quickly, and the signal-to-background ratio is clearer;
(3) the hybridization solution can be suitable for hybridization of different probes including DNA, RNA and the like, and the hybridization time can be effectively shortened, the hybridization effect is optimized, and the background is reduced by using the formula of the hybridization solution.
The hybridization solution is suitable for preparing different in situ hybridization probes, such as DNA in situ hybridization probes, RNA in situ hybridization probes and fluorescent in situ hybridization probes.
The present invention will be described in further detail with reference to specific examples.
The first embodiment is as follows: kappa and Lambda in-situ hybridization detection kit
1. Hybridization solution preparation (50 mL):
(1) weighing 1.25g of sodium fluoride, and adding into a 50mL centrifuge tube;
(2) adding 2.5mL of 1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH7.0 into a 50mL centrifuge tube;
(3) adding 5mL of 20 XSSC solution into a 50mL centrifuge tube;
(4) 0.1mL of 0.5M EDTA solution was added to a 50mL centrifuge tube;
(5) adding 1mL of 50 XDenhardts solution into a 50mL centrifuge tube;
(6) weighing 2.5g of dextran sulfate, adding the dextran sulfate into a 50mL centrifuge tube, fully shaking and uniformly mixing, and centrifuging at 1000rpm for 5 minutes;
(7) adding salmon sperm DNA with final concentration of 0.1 mg/mL;
(8) the volume of ultrapure water added is 50 mL.
Finally, the Kappa and Lambda probes were diluted with the hybridization solution to a final concentration of 1ng/uL, respectively.
2. Experimental methods
2.1 slicing treatment: tissue sections were baked at 60-65 ℃ for more than 60 minutes (positive and negative pairs of photographs were suggested for each experiment).
2.2 dewaxing and hydration: soaking the tissue slices in dewaxing solution for 5 minutes, and repeating for 3 times; soaking in 100% ethanol for 2 min, and repeating for 1 time; the PBS wash was soaked for 2 minutes.
2.3 Add appropriate amount of blocking agent working solution drop by drop per section, to cover the whole tissue, incubate 5 minutes at room temperature. Rinsing with pure water for 30 seconds.
2.4 digestive enzyme digestion: and (4) spin-drying the slices, then sucking the liquid on the edges of the tissue blocks by using absorbent paper, and scribing a water blocking area along the edges of the tissue slices by using an immunohistochemical pen. The sections were placed in a wet box, digestive enzymes (about 50. mu.L, increasing or decreasing depending on the tissue size) were added dropwise, and incubated in an incubator at 37 ℃ for 5-15 minutes (10 minutes were recommended, depending on the tissue type and the section thickness). The PBS wash was soaked for 2 minutes.
2.5 an appropriate amount of Kappa or Lambda chain probe (about 10. mu.L, depending on the tissue size) was added dropwise to each section, and the sections were covered with a glass slide (the sections were dehydrated with 100% ethanol and air-dried before adding the probe dropwise).
2.6 putting the slices into a wet box, and putting the wet box into a constant temperature box at 50 ℃ for 90 minutes to perform denaturation and hybridization; alternatively, denaturation and hybridization were carried out overnight at 37 ℃.
2.7PBS sections were soaked at room temperature, carefully remove the coverslips and continue soaking for 4X 3 minutes.
2.8 spin off the wash, add appropriate primary antibody (about 50. mu.L, increasing or decreasing depending on the tissue size) drop wise, incubate for 30 minutes at room temperature, and soak in PBS for 3X 3 minutes.
2.9 the wash solution was spun off, an appropriate amount of an immunochromatography reagent-A solution (blocking agent, about 50. mu.L, increased or decreased depending on the size of the tissue) was added dropwise, incubated at room temperature for 20 minutes, and soaked in PBS for 3X 3 minutes.
2.10 the wash solution was spun off, an appropriate amount of the immuno-chromogenic reagent-B solution (polymer, about 50. mu.L, increasing or decreasing depending on the tissue size) was added dropwise, incubated at room temperature for 20 minutes, and soaked in PBS for 3X 3 minutes.
2.11 throwing away the cleaning solution, dripping a proper amount of DAB coloration solution to completely cover the specimen, and dyeing for 5 minutes at room temperature (the coloration condition can be observed under a microscope so as to properly adjust the dyeing time).
2.12 counterstaining: rinsing with pure water for 30 seconds, spin-drying, dripping appropriate hematoxylin staining solution to completely cover the specimen, and staining for 3 minutes at room temperature.
2.13 washing with differentiation solution (hydrochloric acid alcohol solution) for 20-30 seconds to remove excess hematoxylin, rinsing with pure water for 30 seconds, and soaking in PBS for 3 minutes to turn blue.
2.14 dehydration, transparency, mounting: soaking the slices in gradient alcohol (the concentration is 75%, 95%, 100% and 100% from low to high) for 2 minutes respectively; and sealing the slices by using the fragrance sealing tablets, and placing the sealed slices in a airing plate.
3. Application example 1
Application example one shows that in the fluorine-containing ion hybridization solution, formamide is not contained, and 2.5% of sodium fluoride is added. When the Kappa probe prepared by the hybridization solution is used for in situ hybridization, a temperature is used for hybridization and denaturation, the signal intensity of the fast hybridization at 50 ℃ for 90 minutes is shown in FIG. 1, and the signal intensity of the overnight hybridization at 37 ℃ is shown in FIG. 2. When the Lambda probe prepared from the hybridization solution is used for in situ hybridization, a temperature is used for hybridization and denaturation, the signal intensity of the rapid hybridization at 50 ℃ for 90 minutes is shown in FIG. 3, and the signal intensity of the overnight hybridization at 37 ℃ is shown in FIG. 4. As can be seen from FIGS. 1-4, when the Kappa and Lambda probes prepared from the hybridization solution are used for in situ hybridization, a single temperature is used for both hybridization and denaturation, and the signal intensity of overnight hybridization at 37 ℃ can be achieved by rapid hybridization at 50 ℃ for 90 minutes. Therefore, the hybridization solution has the advantages that the denaturation temperature is reduced, and the hybridization temperature is increased, so that the whole process can quickly reach the due sensitivity only by one temperature.
Example two: HER2 fluorescent in situ hybridization
1. Hybridization solution preparation (50 mL):
(1) weighing 1.25g of sodium fluoride, and adding into a 50mL centrifuge tube;
(2) adding 2.5mL of 1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH7.0 into a 50mL centrifuge tube;
(3) adding 5mL of 20 XSSC solution into a 50mL centrifuge tube;
(4) adding 0.1mL of 0.5M EDTA solution into a 50mL centrifuge tube;
(5) adding 1mL of 50 XDenhardts solution into a 50mL centrifuge tube;
(6) weighing 5g of dextran sulfate, adding the dextran sulfate into a 50mL centrifuge tube, fully shaking and uniformly mixing, and centrifuging at 1000rpm for 5 minutes;
(7) adding salmon sperm DNA with final concentration of 0.2 mg/mL;
(8) the volume of the added ultrapure water is 50 mL.
Finally, hybridization solution is used for preparing HER2 probes, including a final concentration of 10ng/uL HER2 green fluorescent probe, a final concentration of 1.5ng/uL CEN17 orange fluorescent probe and a final concentration of 1mg/mL human Cot-1 DNA.
2. Experimental methods
2.1 the slides were immersed in xylene (or dewaxed solution) for 3X 10 minutes.
2.2 the slide was immersed in 100%, 100%, 95% and 75% ethanol for 5 minutes, and washed with pure water for 2X 2 minutes.
2.3 preheating the citric acid repair solution to 80 +/-2 ℃ in advance, and putting the slide into the citric acid repair solution to incubate for 30 minutes.
2.4 submerge the slide in purified water for 2X 2 minutes, remove the slide from the purified water, and wipe the excess water from the slide.
2.5 sections were placed in a wet box, digestive enzyme solution was added dropwise, and incubated in a 37 ℃ incubator for 15 minutes. The rinse (2 XSSC) was soaked for 5 minutes. Rinse with deionized water for 1 minute.
2.6, dehydration: 75%, 95%, 100% ethanol solution in turn for 1 minute, air-dried the slices.
2.7 the HER2 probe was removed from the refrigerator and placed at room temperature (18-25 ℃ C.) in the dark. And (5) instantly centrifuging before opening the cover. Pipette 10. mu.L of the probe into each sample. Cover slips after ensuring no air bubbles on slides. The cover glass was sealed with a rubber adhesive.
2.8 placing the slide into a hybridization instrument, and denaturing and hybridizing at 66 ℃ for 2-3 hours or at 50 ℃ overnight.
2.9 carefully remove the rubber seal. The slide was placed in pre-heated wash buffer (2 XSSC, containing 0.1% NP-40) at 37 ℃ and soaked for 1-3 minutes, with the cover slip carefully removed.
2.10 the slides were washed again with pre-heated 37 ℃ wash buffer for 2X 5 minutes.
2.11 put the slide into 75%, 95%, 100% ethanol solution for 1 min each in turn, and dry the sample away from light.
2.12 drops of 25. mu.L of DAPI counterstain were applied to the sliced tissue, covered with a coverslip to avoid air bubbles, and incubated for 15 minutes in the dark. And (4) observing under a fluorescence microscope.
3. Application example two
Application example two shows that the hybridization solution containing fluoride ions does not contain formamide, and 2.5% of sodium fluoride is added. When the HER2 fluorescent in situ hybridization probe prepared by the hybridization solution is used for in situ hybridization, a temperature is used for hybridization and denaturation, the signal intensity of the probe for rapid hybridization at 66 ℃ for 2 hours is shown in figure 5, and the signal intensity of the probe for hybridization at 50 ℃ overnight is shown in figure 6. As can be seen from FIGS. 5-6, when the HER2 fluorescent in situ hybridization probe prepared from the hybridization solution is used for in situ hybridization, one temperature is used for hybridization and denaturation, and the signal intensity of rapid hybridization at 66 ℃ for 2 hours and overnight hybridization at 50 ℃ are very clear and obvious without non-specific background. Therefore, the hybridization solution has the advantages that the denaturation temperature is reduced, and the hybridization temperature is increased, so that the whole process can quickly reach the due sensitivity only by one temperature.
In conclusion, the hybridization solution DNA in situ hybridization probe, RNA in situ hybridization probe and fluorescence in situ hybridization probe for in situ hybridization provided by the invention can reduce the denaturation temperature and simultaneously increase the hybridization temperature, so that the whole process can quickly reach the due sensitivity only by one temperature.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (6)
1. A hybridization solution for in situ hybridization, characterized in that the hybridization solution consists of the following components in final concentrations: formamide with the volume percentage concentration of not more than 2 percent, fluoride with the weight volume percentage concentration of 2.5 percent, SSC with the concentration of 2-4 times, EDTA with the concentration of 1-10mM, Denhardts solution with the concentration of 1-5 times, dextran sulfate with the weight volume percentage concentration of 5-10 percent, salmon sperm DNA with the concentration of 0.1-1mg/mL, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution or Tris-HCl buffer solution with the concentration of 50-100mM and the pH value of 7.0 and ultrapure water.
2. The hybridization solution for in situ hybridization according to claim 1, wherein said fluoride is at least one of sodium fluoride, potassium fluoride and ammonium fluoride.
3. An in situ hybridization assay kit comprising an in situ hybridization assay probe, water and the hybridization solution for in situ hybridization according to any one of claims 1 to 2.
4. The in situ hybridization detection kit according to claim 3, wherein the final concentration of the in situ hybridization detection probe is 1ng/μ L.
5. A method for preparing a hybridization solution for in situ hybridization according to any one of claims 1 to 2, comprising the steps of:
(1) mixing 1.25g of sodium fluoride, 2.5mL of 1M disodium hydrogenphosphate-sodium dihydrogenphosphate buffer, 5mL of 20-fold SSC solution, 0.1mL of 0.5M EDTA solution, 1mL of 50-fold Denhardts solution and 2.5g of dextran sulfate, wherein the pH of the disodium hydrogenphosphate-sodium dihydrogenphosphate buffer is 7.0;
(2) carrying out centrifugal treatment on the mixed solution obtained in the step (1);
(3) salmon sperm DNA was added to a final concentration of 0.1mg/mL, followed by addition of ultrapure water to a volume of 50 mL.
6. The method for preparing hybridization solution for in situ hybridization according to claim 5, wherein the centrifugation process in the step (2) is specifically: centrifuge at 1000rpm for 5 minutes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5750340A (en) * | 1995-04-07 | 1998-05-12 | University Of New Mexico | In situ hybridization solution and process |
| WO2010097655A1 (en) * | 2009-02-26 | 2010-09-02 | Dako Denmark A/S | Compositions and methods for rna hybridization applications |
| CN102046808A (en) * | 2008-05-27 | 2011-05-04 | 丹麦达科有限公司 | Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5750340A (en) * | 1995-04-07 | 1998-05-12 | University Of New Mexico | In situ hybridization solution and process |
| CN102046808A (en) * | 2008-05-27 | 2011-05-04 | 丹麦达科有限公司 | Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers |
| WO2010097655A1 (en) * | 2009-02-26 | 2010-09-02 | Dako Denmark A/S | Compositions and methods for rna hybridization applications |
Non-Patent Citations (2)
| Title |
|---|
| Reduced formamide content and hybridization temperature results in increased non-radioactive mRNA in situ hybridization signals;Alexander Berndt等;《Acta Histochemica》;19960430;第98卷(第2期);全文 * |
| 氟化物对不同类型DNA紫外吸收光谱的影响;高希宝等;《癌变畸变突变》;19980130(第01期);全文 * |
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