CN114058682A - In-situ hybridization buffer solution for anti-counterfeiting and application thereof - Google Patents
In-situ hybridization buffer solution for anti-counterfeiting and application thereof Download PDFInfo
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Abstract
The invention discloses an in-situ hybridization buffer solution for anti-counterfeiting and application thereof. The buffer solution is a mixed solution consisting of an anti-quencher n-propyl gate (NPG), a fluorescent dye PerCP-eFluor 710, a surfactant Tween 80 and Bovine Serum Albumin (BSA), and is prepared from an SSC buffer solution. The final concentration of the anti-quencher NPG is 0.5-12mM, the final concentration of the fluorescent dye PerCP-eFluor 710 is 0.2Mm-2mM, the final concentration of the surfactant Tween 80 is 0.05-1.0%, and the final concentration of Bovine Serum Albumin (BSA) is 1-20 mg/ml. The invention also comprises an operation method for carrying out fluorescence in situ hybridization by using the in situ hybridization buffer solution and an interpretation method for carrying out fluorescence in situ hybridization results by using an AI program with an interpretation anti-counterfeiting function. The method can solve the problem of incorrect interpretation of the interpretation staining result of AI software caused by the use of non-AI corresponding in situ hybridization reagent.
Description
Technical Field
The invention relates to the technical field of chromosome detection, in particular to an in-situ hybridization buffer solution for anti-counterfeiting and application thereof.
Background
In situ hybridization is a detection technique in which labeled exogenous nucleic acid (DNA or RNA) is used as a probe (probe) according to the principle of base pairing of nucleic acid molecules, the labeled exogenous nucleic acid (DNA or RNA) is hybridized with DNA which is denatured on a chromosome and then forms a single strand, and the single strand is recombined to combine into a specific nucleic acid hybrid molecule, and then the position of the exogenous nucleic acid (probe) on the chromosome is directly displayed through detection of the label. The morphology and distribution of cells in stained cells or organelles, or the localization of probe-bound DNA or RNA molecules in chromosomal species can be determined by in situ hybridization. Compared with other chromosome detection methods, the in situ hybridization has the characteristics of high speed, strong detection signal, high hybridization specificity, multiple staining and the like. At present, the technology is widely applied to the fields of animal and plant genome research, chromosome fine structure variation analysis, virus infection analysis, human prenatal diagnosis, tumor genetics, genome evolution research, environmental bacteria sample analysis and the like.
However, the principle of in situ hybridization is complex, the overall steps of the experiment are more, the period is longer, and multiple factors can cause the failure of the hybridization experiment. Moreover, since the interpretation of in situ hybridization depends on the counting of positive cells and negative cells by a pathologist or a technician, different technicians may have different interpretation results for the same result, resulting in wrong interpretation and higher misdiagnosis rate. Currently, the pathological AI company mainly based on Dingjia actively introduces AI technology to assist pathological diagnosis related to various pathological technologies, such as in situ hybridization and the like. However, since the trained AI program and the corresponding reagent need to be completely bound to make an accurate diagnosis, in the actual operation of the pathology department, in-situ hybridization experiments using non-AI corresponding reagents of other brands are often performed, and false interpretation occurs when the staining result is interpreted using AI. Since the staining colors (blue, red and green) of different brands and in-situ hybridization reagents (including fluorescent labeling colors) on tissues are completely consistent, the AI interpretation program cannot distinguish whether the reagents used in the experiment belong to the corresponding reagents according to the staining conditions. This situation will further lead to a continuously high misdiagnosis rate of tumor diagnosis for the patient.
Disclosure of Invention
The invention aims to provide an in-situ hybridization buffer solution for anti-counterfeiting and application thereof, which can solve the problem that an AI software interpretation staining result is wrongly interpreted due to the use of a non-AI corresponding in-situ hybridization reagent.
In order to achieve the purpose, the invention provides the following technical scheme: an in-situ hybridization buffer solution for anti-counterfeiting comprises a mixed solution consisting of an anti-quencher NPG, a PerCP-eFluor 710 fluorescent dye, a Tween 80 surfactant, bovine serum albumin and an SSC buffer solution;
the final concentration of the anti-quencher NPG is 0.5-12mM, the final concentration of the PerCP-eFluor 710 fluorescent dye is 0.2Mm-2mM, the final concentration of the Tween 8 surfactant 0 is 0.05-1.0%, and the final concentration of the bovine serum albumin is 1-20 mg/ml.
The preparation method of the in situ hybridization buffer solution comprises the following steps:
s1, finishing the whole preparation process in aseptic operation;
s2, taking 500ml SSC buffer solution, adding 2.5g of bovine serum albumin into the buffer solution, wherein the final concentration of the bovine serum albumin is 5mg/ml, and fully mixing for 5min by using a vortex mixer;
s3, pouring the mixed diluent into a light-proof brown bottle, adding 0.424g of anti-quencher NPG powder, wherein the final concentration of the anti-quencher NPG is 2mM, and fully mixing for 5min by using a vortex mixer;
s4, slowly adding 100ul of PerCP-eFluor 710 fluorescent dye concentrated solution into the brown bottle, wherein the final concentration of the PerCP-eFluor 710 fluorescent dye is 0.5mM, and then slightly reversing and mixing evenly for 3-5 times;
and S5, finally adding 1ml of Tween 80 with the final concentration of 0.2%, and slowly mixing the mixture on a blood mixing instrument for 30min to obtain the in-situ hybridization buffer solution with the fluorescent group and capable of AI interpretation anti-counterfeiting.
The application of in-situ hybridization buffer solution for anti-counterfeiting comprises an operation method for performing fluorescence in-situ hybridization by using the in-situ hybridization buffer solution and an interpretation method for performing fluorescence in-situ hybridization results by using an AI program with an interpretation anti-counterfeiting function.
The operation method for performing fluorescence in situ hybridization by using the in situ hybridization buffer solution comprises the following steps:
c1, continuously slicing 2 lung non-small cell lung cancer tissues from the same source, and baking the slices in a thermostat at 65 ℃ for one night;
c2, taking out the slices the next day, sequentially putting all the slices into fresh dimethylbenzene, and soaking for 10 minutes; soaking in anhydrous ethanol for 10 min; soaking in 90% ethanol for 3 min; soaking the slices in 70% ethanol for 3 min; placing the slices in purified water, soaking for 3 minutes, taking out the slices, and throwing off excessive water;
c3, taking out the slices, putting the slices into boiling purified water at the temperature of 100 ℃, enabling water to permeate the slices, and maintaining the temperature for 25 minutes;
c4, taking out the slices, and airing at room temperature; horizontally placing the section with the front side facing upwards, dripping 200ul of pepsin working solution into the tissue area, and digesting for 20 minutes;
c5, throwing off excessive liquid, and putting slices into 2 XSSC buffer solution at room temperature for 3 minutes; taking out the slices, and sequentially adding 70%, 90% and 100% gradient ethanol for dehydration for 2 minutes respectively;
c6, taking out the slices, and airing at room temperature;
c7, dripping 10ul of the working solution of the fluorescence in-situ hybridization reagent into the hybridization area on one slice, quickly covering the slide glass, and slightly pressing to uniformly distribute the hybridization reagent so as to avoid generating bubbles; then, dripping the working liquid of the common in-situ hybridization reagent into the hybridization area on the other slice, and then, completely consistent other steps;
c8, sealing the slide along the edge of the cover glass by using rubber glue, and completely covering the contact part of the cover glass and the slide glass;
c9, putting the slices into a hybridization instrument, wetting the humidity strip of the in situ hybridization instrument, inserting the wet strip, covering the upper cover of the hybridization instrument, setting the program, and performing denaturation at 85 ℃ for 5 minutes and hybridization at 37 ℃ for 18 hours.
C10, preparing 100ml of 2 XSSC buffer incubated at 37 ℃ and 100ml of 2 XSSC buffer containing 0.1% NP40 in advance before the experiment on the next day; taking out the slices in the hybridization instrument, slightly tearing off the rubber, removing the cover glass, and putting the slices into a 2 XSSC buffer solution at 37 ℃ for incubation for 10 minutes;
c11, taking out the section, and then putting the section into 2 XSSC buffer solution containing 0.1% NP40 at 37 ℃ for incubation for 10 minutes;
c12, taking out the slices, and incubating in 70% ethanol at room temperature for 3 minutes; taking out the slices, and putting the slices into a dark box for airing;
c13, dripping 10ul of DAPI compound dye solution on the cover glass, reversely slicing to enable the cover glass to be respectively contacted with the target area of the glass slide, and slightly pressing under counter pressure to avoid generating bubbles;
c14, storing in dark and observing signals.
The interpretation method of the fluorescence in situ hybridization result by using the AI program with the interpretation anti-counterfeiting function comprises the following steps:
m1, placing lung small cell lung cancer slices made by the in-situ hybridization anti-counterfeiting buffer solution on an objective table of a fluorescence microscope connected with an AI interpretation software module, opening intelligent pathology AI interpretation software, after the software and the microscope are successfully connected, opening a fluorescence light source of the fluorescence microscope, exciting laser with the wavelength of 633nm emitted by the light source through a grating, enabling fluorescence to enter a camera through the slices on the objective table, analyzing fluorescence signals with the wavelength of 710nm by the intelligent pathology AI interpretation software, automatically performing watershed algorithm segmentation and tracking algorithm clustering on a red channel only after a digital image is calibrated by automatic background gray scale, performing average optical density calculation on a red bright part, and if the average optical density reaches 130, popping prompt information by the software: the slice is qualified and can be used for AI interpretation; namely, the reagent used by the section is qualified in situ hybridization reagent which can be interpreted by AI;
m2, section software passing reagent verification can automatically close an excitation light source with the wavelength of 633nm, open an excitation light source with the wavelength of 496nm, search and interpret a green fluorescence signal of a proper positive cell, and take a picture at the wavelength after finding an area with proper intensity and density; then opening a 552nm wavelength excitation light source, observing the condition of a red fluorescence signal of the positive cell, and taking a picture at the wavelength after the condition is confirmed to be correct; and opening an excitation light source with the wavelength of 367nm, observing the condition of the blue fluorescence signal of the DAPI cell nucleus, and taking a picture at the wavelength after confirming that no error exists.
M3, automatically synthesizing the 3 taken fluorescence pictures in intelligent pathological AI interpretation software, and selecting different modules by an operator according to different hybridized genes to perform subsequent fluorescence in-situ hybridization interpretation;
m4, placing another lung non-small cell lung cancer slice made of a common in-situ hybridization reagent working solution on an objective table, carrying out reagent verification according to the same method, acquiring the value of the fluorescence signal intensity of a red channel of a digital image under the excitation of laser with a wavelength of 633nm, processing after automatic background gray calibration, carrying out automatic watershed algorithm segmentation and tracking algorithm clustering on the red channel only, wherein the average optical density of the fluorescence signal corresponding to the wavelength of 710nm is less than 130, and intelligent pathology AI interpretation software pops out prompt information: unqualified slices are cut, and a qualified AI in-situ hybridization corresponding reagent is used; the subsequent section cannot be interpreted through intelligent pathology AI software;
m5, shooting the section at 496nm, 552nm and 367nm by using a common fluorescence microscope without intelligent pathology AI software, and comparing the section with the staining effect of an AI in situ hybridization buffer solution; as a result, the fluorescence intensity and the area of the diluted two different in situ hybridization buffers are basically consistent in a plurality of wave bands, and the performance is not obviously changed.
Compared with the prior art, the invention has the following beneficial effects:
after the four components are mixed, a fluorescent signal with the wavelength of 710nm, uniform distribution of fluorescent groups, stable intensity and difficult quenching can be obtained after the four components are excited by exciting light with the wavelength of 633 nm. After a fluorescent signal enters a camera through an objective lens in a microscope, after digital images of the fluorescent signal are subjected to automatic background gray scale calibration, only a red channel is subjected to automatic Watershed (filtered) algorithm segmentation and tracking algorithm (Mean Shift) for clustering, and red bright places (which can be considered as identified fluorescent signals) are subjected to average optical density calculation, if the red bright places reach 130, the reagent system corresponding to the correct AI can be judged, and then the subsequent immunohistochemical automatic interpretation can be carried out. If the value is less than 130, the reagent system corresponding to the incorrect AI is determined, and the AI interpretation software will prompt the problem not to be interpreted. The method can solve the problem of incorrect interpretation of the interpretation staining result of AI software caused by the use of non-AI corresponding in situ hybridization reagent.
Drawings
FIG. 1 is a functional interface screenshot for detecting lung non-small cell lung cancer slices made by a fluorescence in situ hybridization anti-counterfeiting reagent using AI interpretation software;
FIG. 2 is a screenshot of a prompt message interface when the AI interpretation software is used to analyze the slice as qualified;
FIG. 3 is a screenshot of 3 fluorescence pictures taken at 496nm, 552nm and 367nm automatically synthesized by AI interpretation software;
FIG. 4 is a functional interface screenshot for using AI interpretation software to detect lung NSCLC slices prepared from a common in situ hybridization reagent working solution;
FIG. 5 is a screenshot of a prompt message interface when AI interpretation software is used to analyze a section that is unacceptable;
fig. 6 is a screenshot of a composite image of 3 fluorescence pictures taken at 496nm, 552nm and 367nm using a general fluorescence microscope without intelligent pathology AI software.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: an in-situ hybridization buffer solution for anti-counterfeiting is a mixed solution consisting of an anti-quencher n-propyl gate (NPG), a fluorescent dye PerCP-eFluor 710, a surfactant Tween 80 and Bovine Serum Albumin (BSA), and is prepared from an SSC buffer solution. The final concentration of the anti-quencher NPG is 0.5-12mM, the final concentration of the fluorescent dye PerCP-eFluor 710 is 0.2Mm-2mM, the final concentration of the surfactant Tween 80 is 0.05-1.0%, and the final concentration of Bovine Serum Albumin (BSA) is 1-20 mg/ml.
The fluorescent dye can generate nonspecific combination with tissue slices at room temperature, can be uniformly distributed on the slice tissues, and does not influence the subsequent related dyeing of fluorescence in situ hybridization specificity due to the nonspecific combination and the larger difference between the light-emitting waveband and the normal fluorescence in situ hybridization excitation light waveband. The anti-quenching agent NPG is a nontoxic antioxidant, can provide free hydrogen atoms to combine with free radicals generated by oxidation so as to achieve the effect of resisting oxidation, and can effectively prevent the quenching of the fluorescent dye. Tween 80 is a polyol-type nonionic surfactant, so that fluorescent dye can be distributed on the sliced tissue more uniformly, and the overall stability of the buffer solution is improved. BSA is a protein with stable performance, and BSA with higher concentration can improve the stability of the hybridization probe and slow down the degradation speed of the probe. After the four components are mixed, a fluorescent signal with the wavelength of 710nm, uniform distribution of fluorescent groups, stable intensity and difficult quenching can be obtained after the four components are excited by exciting light with the wavelength of 633 nm. After a fluorescent signal enters a camera through an objective lens in a microscope, after digital images of the fluorescent signal are subjected to automatic background gray scale calibration, only a red channel is subjected to automatic Watershed (filtered) algorithm segmentation and tracking algorithm (Mean Shift) for clustering, and red bright places (which can be considered as identified fluorescent signals) are subjected to average optical density calculation, if the red bright places reach 130, the reagent system corresponding to the correct AI can be judged, and then the subsequent immunohistochemical automatic interpretation can be carried out. If the value is less than 130, the reagent system corresponding to the incorrect AI is determined, and the AI interpretation software will prompt the problem not to be interpreted.
Preparing in-situ hybridization reagent buffer solution with fluorescent dye, and specifically comprising the following operation steps:
s1, finishing the whole preparation process in aseptic operation;
s2, taking 500ml SSC buffer solution, adding 2.5g of bovine serum albumin into the buffer solution, wherein the final concentration of the bovine serum albumin is 5mg/ml, and fully mixing for 5min by using a vortex mixer;
s3, pouring the mixed diluent into a light-proof brown bottle, adding 0.424g of anti-quencher NPG powder, wherein the final concentration of the anti-quencher NPG is 2mM, and fully mixing for 5min by using a vortex mixer;
s4, slowly adding 100ul of PerCP-eFluor 710 fluorescent dye concentrated solution into the brown bottle, wherein the final concentration of the PerCP-eFluor 710 fluorescent dye is 0.5mM, and then slightly reversing and mixing evenly for 3-5 times;
and S5, finally adding 1ml of Tween 80 with the final concentration of 0.2%, and slowly mixing the mixture on a blood mixing instrument for 30min to obtain the in-situ hybridization buffer solution with the fluorescent group and capable of AI interpretation anti-counterfeiting. .
The operation method for performing fluorescence in situ hybridization by using the in situ hybridization buffer solution comprises the following steps:
meanwhile, a fluorescence in situ hybridization reagent of common buffer solution is used for contrast;
taking an EML4/ALK fusion gene detection reagent as an example in the following experimental process, firstly, using a probe concentrated solution and an in situ hybridization reagent buffer solution prepared in the technical scheme 1 according to the proportion of 1: 5, and obtaining diluted in situ hybridization reagent working solution with fluorescence for fluorescence in situ hybridization experiments. Simultaneously using SSC buffer solution according to the weight ratio of 1: 5, diluting the probe concentrated solution to obtain the common in-situ hybridization reagent working solution.
C1, continuously slicing 2 lung non-small cell lung cancer tissues from the same source, and baking the slices in a thermostat at 65 ℃ for one night;
c2, taking out the slices the next day, sequentially putting all the slices into fresh dimethylbenzene, and soaking for 10 minutes; soaking in anhydrous ethanol for 10 min; soaking in 90% ethanol for 3 min; soaking the slices in 70% ethanol for 3 min; placing the slices in purified water, soaking for 3 minutes, taking out the slices, and throwing off excessive water;
c3, taking out the slices, putting the slices into boiling purified water at the temperature of 100 ℃, enabling water to permeate the slices, and maintaining the temperature for 25 minutes;
c4, taking out the slices, and airing at room temperature; horizontally placing the section with the front side facing upwards, dripping 200ul of pepsin working solution into the tissue area, and digesting for 20 minutes;
c5, throwing off excessive liquid, and putting slices into 2 XSSC buffer solution at room temperature for 3 minutes; taking out the slices, and sequentially adding 70%, 90% and 100% gradient ethanol for dehydration for 2 minutes respectively;
c6, taking out the slices, and airing at room temperature;
c7, dripping 10ul of the working solution of the fluorescence in-situ hybridization reagent into the hybridization area on one slice, quickly covering the slide glass, and slightly pressing to uniformly distribute the hybridization reagent so as to avoid generating bubbles; then, dripping the working liquid of the common in-situ hybridization reagent into the hybridization area on the other slice, and then, completely consistent other steps;
c8, sealing the slide along the edge of the cover glass by using rubber glue, and completely covering the contact part of the cover glass and the slide glass;
c9, putting the slices into a hybridization instrument, wetting the humidity strip of the in situ hybridization instrument, inserting the wet strip, covering the upper cover of the hybridization instrument, setting the program, and performing denaturation at 85 ℃ for 5 minutes and hybridization at 37 ℃ for 18 hours.
C10, preparing 100ml of 2 XSSC buffer incubated at 37 ℃ and 100ml of 2 XSSC buffer containing 0.1% NP40 in advance before the experiment on the next day; taking out the slices in the hybridization instrument, slightly tearing off the rubber, removing the cover glass, and putting the slices into a 2 XSSC buffer solution at 37 ℃ for incubation for 10 minutes;
c11, taking out the section, and then putting the section into 2 XSSC buffer solution containing 0.1% NP40 at 37 ℃ for incubation for 10 minutes;
c12, taking out the slices, and incubating in 70% ethanol at room temperature for 3 minutes; taking out the slices, and putting the slices into a dark box for airing;
c13, dripping 10ul of DAPI compound dye solution on the cover glass, reversely slicing to enable the cover glass to be respectively contacted with the target area of the glass slide, and slightly pressing under counter pressure to avoid generating bubbles;
c14, storing in dark and observing signals.
The interpretation method of the fluorescence in situ hybridization result by using the AI program with the interpretation anti-counterfeiting function is as follows (using a non-anti-counterfeiting reagent for comparison):
m1, firstly, placing the lung non-small cell lung cancer slice made of the fluorescence in-situ hybridization anti-counterfeiting reagent on an objective table of a fluorescence microscope connected with an AI interpretation software module, opening intelligent pathology AI interpretation software, clicking 'start processing' in a 'reagent verification' menu item after the software and the microscope are successfully connected (see figure 1), opening a fluorescence light source by the fluorescence microscope, exciting the light source by laser with the wavelength of 633nm through a grating, enabling the fluorescence to enter a camera through the slice on the objective table, analyzing a fluorescence signal with the wavelength of 710nm by the intelligent pathology AI interpretation software, carrying out automatic Watershed (filtered) algorithm segmentation and tracking algorithm (Mean Shift) on a red channel after the digital image is subjected to automatic background gray calibration, and carrying out average optical density calculation on a red bright part (which can be considered as an identified fluorescence signal), if 130 is reached, the software pops up a prompt: the sections were qualified and available for AI interpretation (see fig. 2). That is, the reagent used for the section is a qualified in situ hybridization reagent that can be interpreted as an AI.
M2, section software passing reagent verification can automatically close the excitation light source with 633nm wavelength, open the excitation light source with 496nm wavelength, search and interpret the green fluorescence signal of the appropriate positive cell, and take a picture at the wavelength after finding the area with appropriate intensity and density. Then, an excitation light source with the wavelength of 552nm is turned on, the condition of a red fluorescence signal of the positive cell is observed, and a picture is taken at the wavelength after the condition is confirmed to be correct. And opening an excitation light source with the wavelength of 367nm, observing the condition of the blue fluorescence signal of the DAPI cell nucleus, and taking a picture at the wavelength after confirming that no error exists.
M3, automatically synthesizing the 3 taken fluorescence pictures in intelligent pathology AI interpretation software (see figure 3), and selecting different modules by an operator according to different hybridized genes to perform subsequent fluorescence in situ hybridization interpretation.
M4, placing another lung non-small cell lung cancer slice made of a common in-situ hybridization reagent working solution on an object stage, carrying out reagent verification according to the same method, acquiring the value of the fluorescence signal intensity of a red channel of a digital image under the excitation of laser with a wavelength of 633nm, processing after automatic background gray calibration, carrying out automatic Watershed (Watershed) algorithm segmentation and tracking algorithm (Mean Shift) clustering on the red channel only, wherein the Mean optical density of the fluorescence signal corresponding to the wavelength of 710nm is less than 130 (see figure 4), and intelligent pathology AI interpretation software pops up prompt information: the section was rejected and please use the qualified AI in situ hybridization assay (see FIG. 5). The subsequent section cannot be interpreted by intelligent pathology AI software.
M5, the section was photographed at 496nm, 552nm and 367nm using a general fluorescence microscope without intelligent pathology AI software (see FIG. 6) for comparison with the staining effect of AI in situ hybridization reagents. As a result, the fluorescence intensity and the area of the diluted two different in situ hybridization buffers are basically consistent in a plurality of wave bands, and the performance is not obviously changed.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. An in-situ hybridization buffer solution for anti-counterfeiting is characterized by comprising a mixed solution consisting of an anti-quencher NPG, a PerCP-eFluor 710 fluorescent dye, a Tween 80 surfactant, bovine serum albumin and an SSC buffer solution;
the final concentration of the anti-quencher NPG is 0.5-12mM, the final concentration of the PerCP-eFluor 710 fluorescent dye is 0.2Mm-2mM, the final concentration of the Tween 8 surfactant 0 is 0.05-1.0%, and the final concentration of the bovine serum albumin is 1-20 mg/ml.
2. The in situ hybridization buffer solution for anti-counterfeiting according to claim 1, wherein the preparation method of the in situ hybridization buffer solution is as follows:
s1, finishing the whole preparation process in aseptic operation;
s2, taking 500ml SSC buffer solution, adding 2.5g of bovine serum albumin into the buffer solution, wherein the final concentration of the bovine serum albumin is 5mg/ml, and fully mixing for 5min by using a vortex mixer;
s3, pouring the mixed diluent into a light-proof brown bottle, adding 0.424g of anti-quencher NPG powder, wherein the final concentration of the anti-quencher NPG is 2mM, and fully mixing for 5min by using a vortex mixer;
s4, slowly adding 100ul of PerCP-eFluor 710 fluorescent dye concentrated solution into the brown bottle, wherein the final concentration of the PerCP-eFluor 710 fluorescent dye is 0.5mM, and then slightly reversing and mixing evenly for 3-5 times;
and S5, finally adding 1ml of Tween 80 with the final concentration of 0.2%, and slowly mixing the mixture on a blood mixing instrument for 30min to obtain the in-situ hybridization buffer solution with the fluorescent group and capable of AI interpretation anti-counterfeiting.
3. The use of the in situ hybridization buffer according to claim 2 for anti-counterfeiting, wherein the in situ hybridization buffer comprises: comprises an operation method for carrying out fluorescence in situ hybridization by using the in situ hybridization buffer solution and an interpretation method for carrying out fluorescence in situ hybridization results by using an AI program with an interpretation anti-counterfeiting function.
4. The use of the in situ hybridization buffer according to claim 3 for anti-counterfeiting, wherein the operation method of performing fluorescence in situ hybridization using the in situ hybridization buffer is as follows:
c1, continuously slicing 2 lung non-small cell lung cancer tissues from the same source, and baking the slices in a thermostat at 65 ℃ for one night;
c2, taking out the slices the next day, sequentially putting all the slices into fresh dimethylbenzene, and soaking for 10 minutes; soaking in anhydrous ethanol for 10 min; soaking in 90% ethanol for 3 min; soaking the slices in 70% ethanol for 3 min; placing the slices in purified water, soaking for 3 minutes, taking out the slices, and throwing off excessive water;
c3, taking out the slices, putting the slices into boiling purified water at the temperature of 100 ℃, enabling water to permeate the slices, and maintaining the temperature for 25 minutes;
c4, taking out the slices, and airing at room temperature; horizontally placing the section with the front side facing upwards, dripping 200ul of pepsin working solution into the tissue area, and digesting for 20 minutes;
c5, throwing off excessive liquid, and putting slices into 2 XSSC buffer solution at room temperature for 3 minutes; taking out the slices, and sequentially adding 70%, 90% and 100% gradient ethanol for dehydration for 2 minutes respectively;
c6, taking out the slices, and airing at room temperature;
c7, dripping 10ul of the working solution of the fluorescence in-situ hybridization reagent into the hybridization area on one slice, quickly covering the slide glass, and slightly pressing to uniformly distribute the hybridization reagent so as to avoid generating bubbles; then, dripping the working liquid of the common in-situ hybridization reagent into the hybridization area on the other slice, and then, completely consistent other steps;
c8, sealing the slide along the edge of the cover glass by using rubber glue, and completely covering the contact part of the cover glass and the slide glass;
c9, putting the slices into a hybridization instrument, wetting a humidity strip of the in-situ hybridization instrument, inserting the wet strip, covering an upper cover of the hybridization instrument, setting a program, and performing denaturation at 85 ℃ for 5 minutes and hybridization at 37 ℃ for 18 hours;
c10, preparing 100ml of 2 XSSC buffer incubated at 37 ℃ and 100ml of 2 XSSC buffer containing 0.1% NP40 in advance before the experiment on the next day; taking out the slices in the hybridization instrument, slightly tearing off the rubber, removing the cover glass, and putting the slices into a 2 XSSC buffer solution at 37 ℃ for incubation for 10 minutes;
c11, taking out the section, and then putting the section into 2 XSSC buffer solution containing 0.1% NP40 at 37 ℃ for incubation for 10 minutes;
c12, taking out the slices, and incubating in 70% ethanol at room temperature for 3 minutes; taking out the slices, and putting the slices into a dark box for airing;
c13, dripping 10ul of DAPI compound dye solution on the cover glass, reversely slicing to enable the cover glass to be respectively contacted with the target area of the glass slide, and slightly pressing under counter pressure to avoid generating bubbles;
c14, storing in dark and observing signals.
5. The use of the in situ hybridization buffer solution for anti-counterfeiting prevention according to claim 4, wherein the AI program with the anti-counterfeiting interpretation function is used for the interpretation method of the fluorescence in situ hybridization result as follows:
m1, placing lung small cell lung cancer slices made by the in-situ hybridization anti-counterfeiting buffer solution on an objective table of a fluorescence microscope connected with an AI interpretation software module, opening intelligent pathology AI interpretation software, after the software and the microscope are successfully connected, opening a fluorescence light source of the fluorescence microscope, exciting laser with the wavelength of 633nm emitted by the light source through a grating, enabling fluorescence to enter a camera through the slices on the objective table, analyzing fluorescence signals with the wavelength of 710nm by the intelligent pathology AI interpretation software, automatically performing watershed algorithm segmentation and tracking algorithm clustering on a red channel only after a digital image is calibrated by automatic background gray scale, performing average optical density calculation on a red bright part, and if the average optical density reaches 130, popping prompt information by the software: the slice is qualified and can be used for AI interpretation; namely, the reagent used by the section is qualified in situ hybridization reagent which can be interpreted by AI;
m2, section software passing reagent verification can automatically close an excitation light source with the wavelength of 633nm, open an excitation light source with the wavelength of 496nm, search and interpret a green fluorescence signal of a proper positive cell, and take a picture at the wavelength after finding an area with proper intensity and density; then opening a 552nm wavelength excitation light source, observing the condition of a red fluorescence signal of the positive cell, and taking a picture at the wavelength after the condition is confirmed to be correct; opening a 367nm wavelength excitation light source, observing the condition of the DAPI cell nucleus blue fluorescence signal, and taking a picture at the wavelength after confirming no error;
m3, automatically synthesizing the 3 taken fluorescence pictures in intelligent pathological AI interpretation software, and selecting different modules by an operator according to different hybridized genes to perform subsequent fluorescence in-situ hybridization interpretation;
m4, placing another lung non-small cell lung cancer slice made of a common in-situ hybridization reagent working solution on an objective table, carrying out reagent verification according to the same method, acquiring the value of the fluorescence signal intensity of a red channel of a digital image under the excitation of laser with a wavelength of 633nm, processing after automatic background gray calibration, carrying out automatic watershed algorithm segmentation and tracking algorithm clustering on the red channel only, wherein the average optical density of the fluorescence signal corresponding to the wavelength of 710nm is less than 130, and intelligent pathology AI interpretation software pops out prompt information: unqualified slices are cut, and a qualified AI in-situ hybridization corresponding reagent is used; the subsequent section cannot be interpreted through intelligent pathology AI software;
m5, shooting the section at 496nm, 552nm and 367nm by using a common fluorescence microscope without intelligent pathology AI software, and comparing the section with the staining effect of an AI in situ hybridization buffer solution; as a result, the fluorescence intensity and the area of the diluted two different in situ hybridization buffers are basically consistent in a plurality of wave bands, and the performance is not obviously changed.
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