CN115976169A - Pretreatment liquid for FISH detection of paraffin-embedded tissue sample and application thereof - Google Patents

Abstract

The invention discloses a pretreatment liquid for FISH detection of a paraffin-embedded tissue sample and application thereof. The pretreatment solution contains a protective solution and a transparent agent, the protective solution contains ethylene diamine tetraacetic acid, alkali metal chloride and a buffer solution, and the transparent agent contains any one or the combination of at least two of tert-butyl alcohol, polyethylene glycol or a surfactant. The invention develops a brand-new pretreatment liquid formula, utilizes the synergistic cooperation of all components, can effectively aim at old paraffin-embedded tissue samples which are prepared for more than 3 years, promotes the FISH probe to enter cell nuclei, reduces tissue shedding, effectively hybridizes, reduces fluorescence background, is beneficial to FISH signal observation, and can be effectively applied to retrospective research on old samples or research on first paraffin-embedded tissue samples of relapsing patients and the like.

Description

Pretreatment liquid for FISH detection of paraffin-embedded tissue sample and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a pretreatment liquid for FISH detection of paraffin-embedded tissue samples and application thereof.

Background

Conventional paraffin-embedded tissue sections (FFPE) are the most widely used method in conventional histological slide-making techniques, including: (1) fixing tissues: placing the specimen in formalin solution; and (2) paraffin embedding: after formalin fixation, tissue specimens were embedded in paraffin. In pathology, it is often used to study, observe and judge morphological changes in cellular tissues. The formalin-fixed paraffin-embedded tissue is the most common sample preservation mode in the pathology department, the sample preserved by the method is stable, the preservation condition is simple and convenient, the sample can be preserved in hospital files for years to decades, and the formalin-fixed paraffin-embedded tissue is the best material for backtracking clinical samples. However, as the storage time of the sample is prolonged, the nucleic acid and protein of the sample are degraded and the degree of cross-linking is gradually increased.

Fluorescence In Situ Hybridization (FISH) is a clinical application method which can directly research gene changes on the cellular level, can be used for gene localization and state analysis In cells by combining with the FISH method, and is a technology which has very important value In the aspect of diagnosing hereditary diseases and malignant tumors and is generated on the basis of combining cytogenetics and DNA technology. The preparation process of the FISH probe comprises the following steps: nick translation, random primer marking, RNA transcription marking, PCR marking and the like. The labeled probe is denatured and annealed with chromosome under the assistance of hybridization buffer solution at proper temperature, then the excess probe is washed away by proper buffer solution, cell nucleus staining solution such as 4', 6-diamidino-2-phenylindole (DAPI) or Hochest33285 and the like is added for nonspecific DNA staining, and the cell nucleus staining solution can be observed under a microscope after the cell nucleus staining solution is mounted.

Among tumor-related differential diagnosis technologies, FFPE samples are common sample forms of most solid tumors, in clinical practice, samples over 3 years are prepared, and due to slow and continuous formaldehyde fixation, severe fragmentation occurs, nucleic acid easily falls off during the preparation process, which affects the experimental results, the hybridization signal is weak, which is not beneficial to FISH signal observation, and the FFPE samples can hardly be used for FISH technical detection, so that the FISH technical cannot carry out retrospective research on old samples or research on first FFPE samples of relapsed patients, and therefore, development of a pretreatment method for old FFPE sample FISH detection becomes a focus of attention, and CN105018598A discloses a FISH pretreatment method and a pretreatment liquid for FFPE samples, wherein the pretreatment liquid comprises: pretreatment liquid I: at least one of EDTA solution, citric acid buffer solution and sodium thiocyanate solution; pretreatment liquid II: at least one of pontamine sky blue at a concentration of 0.01-0.5% and trypan blue at a concentration of 0.025-1%; pretreatment liquid III: sodium borohydride at a concentration of 0.1-5%, the above method is still difficult to effectively target old FFPE samples.

In conclusion, the development of the pretreatment liquid and the method which can be used for the FISH detection of the old FFPE sample has important significance for the FISH field.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the pretreatment liquid for FISH detection of paraffin-embedded tissue samples and the application thereof, which can effectively carry out pretreatment on old paraffin-embedded tissue samples for more than 3 years, so that the treated samples can effectively carry out FISH detection.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a pretreatment liquid for FISH detection of paraffin-embedded tissue samples, which comprises a protective liquid and a clearing agent, wherein the protective liquid comprises Ethylene Diamine Tetraacetic Acid (EDTA), an alkaline metal chloride and a buffer liquid, and the clearing agent comprises any one or a combination of at least two of tert-butyl alcohol, polyethylene glycol or a surfactant.

According to the invention, a brand-new pretreatment liquid formula is developed, EDTA is used as a crosslinking release buffer component, the alkaline metal chloride provides a cationic environment, the flaking is reduced, the crosslinking is released gently, the buffer provides a buffer environment, the fluctuation of the pH value in the flaking boiling process is reduced, the transparentizing agent dissolves part of lipid and components, the cells become more uniform and transparent, and the components cooperate with each other, so that the FISH probe can be promoted to enter the cell nucleus of an old sample with the preparation time of more than 3 years, the tissue shedding is reduced, the effective hybridization is realized, the fluorescence background is reduced, and the FISH signal observation is facilitated.

Preferably, the alkali metal chloride comprises any one of sodium chloride, potassium chloride or lithium chloride or a combination of at least two thereof.

Preferably, the buffer comprises any one of a Tris-HCl buffer, a potassium dihydrogen phosphate-sodium hydroxide buffer, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, or a barbituric sodium-hydrochloric acid buffer.

Preferably, the polyethylene glycol has an average relative molecular mass of 200 to 1500, including but not limited to 400, 500, 800, 1000 or 1300.

Preferably, the surfactant comprises any one of polyethylene glycol p-isooctyl phenyl ether (triton), dodecyl sulfate, tween or NP40 or a combination of at least two thereof.

Preferably, the triton comprises triton-100 and/or triton-114.

Preferably, the lauryl sulfate salt comprises sodium lauryl sulfate and/or lithium lauryl sulfate.

Preferably, the tween comprises tween-20 and/or tween-80.

Preferably, the concentration of the ethylenediaminetetraacetic acid in the pretreatment liquid is 10-100 mmol/L (mM), including but not limited to 11mmol/L, 12mmol/L, 13mmol/L, 15mmol/L, 20mmol/L, 25mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L, 92mmol/L, 95mmol/L, 96mmol/L, 98mmol/L or 99mmol/L.

Preferably, the concentration of the alkali metal chloride in the pretreatment solution is 50-300 mmol/L, including but not limited to 51mmol/L, 52mmol/L, 55mmol/L, 58mmol/L, 60mmol/L, 70mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L, 260mmol/L, 280mmol/L, 290mmol/L, 292mmol/L, 295mmol/L, 296mmol/L, 298mmol/L or 299mmol/L.

Preferably, the concentration of the buffer salt in the pretreatment solution is 5 to 50mmol/L, including but not limited to 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L, 15mmol/L, 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 42mmol/L, 45mmol/L, 46mmol/L, 47mmol/L, 48mmol/L or 49mmol/L.

Preferably, the mass percentage of the clearing agent in the pretreatment liquid is 0.5 to 10%, including but not limited to 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 7%, 9%, 9.5%, 9.8% or 9.9%.

In a second aspect, the present invention provides a method for preparing the pretreatment liquid for FISH assay of paraffin-embedded tissue samples according to the first aspect, the method comprising:

and mixing the Ethylene Diamine Tetraacetic Acid (EDTA), the alkaline metal chloride, a buffer solution and the clearing agent to obtain the pretreatment solution.

In a third aspect, the invention provides the use of the pretreatment liquid for FISH examination of paraffin-embedded tissue samples according to the first aspect in the preparation of a product for examination of paraffin-embedded tissue samples.

In a fourth aspect, the present invention provides a paraffin-embedded tissue sample pretreatment kit, which comprises the pretreatment solution for FISH detection of paraffin-embedded tissue samples according to the first aspect.

In a fifth aspect, the invention provides a paraffin-embedded tissue sample FISH detection kit, which comprises the pretreatment liquid for FISH detection of paraffin-embedded tissue samples in the first aspect.

Preferably, the kit further comprises FISH detection reagents.

It is understood that FISH detection reagents commonly used in the art are suitable for use in the present invention.

Preferably, the FISH detection reagent comprises protease for in situ hybridization (20160321 number of Guangdong ear mechanical equipment), HER2 gene detection kit (fluorescence in situ hybridization method) national mechanical standard 20213400138, ROS1 gene disruption detection kit (fluorescence in situ hybridization method) (Guangzhou Anbipin medicine science and technology, inc.) and in situ hybridization blue staining solution (20160322 number of Guangdong ear mechanical equipment).

In a sixth aspect, the invention provides the use of the pretreatment fluid for FISH examination of paraffin-embedded tissue samples according to the first aspect in FISH examination of paraffin-embedded tissue samples.

In a seventh aspect, the present invention provides a pretreatment method for FISH detection of paraffin-embedded tissue samples, the method comprising:

sequentially carrying out slicing treatment, baking treatment and dewaxing treatment on the paraffin-embedded tissue sample to be detected, mixing the dewaxed sample with the pretreatment liquid for FISH detection of the paraffin-embedded tissue sample in the first aspect, carrying out heating treatment, and carrying out enzyme digestion treatment on the heated sample.

Preferably, the heat treatment is at a temperature of 60 to 95 ℃, including but not limited to 61 ℃, 62 ℃, 65 ℃, 68 ℃, 70 ℃, 75 ℃,80 ℃, 85 ℃, 88 ℃, 89 ℃, 92 ℃, 93 ℃ or 94 ℃, for a time of 10 to 2000min, including but not limited to 11min, 12min, 15min, 18min, 20min, 100min, 200min, 500min, 600min, 800min, 1500min, 1800min, 1900min, 1950min, 1960min, 1980min, 1995min, 1996min, 1997min or 1999min.

Preferably, the tool enzyme for the enzymatic digestion treatment comprises any one of proteinase K, pepsin or trypsin, or a combination of at least two of them.

Preferably, the enzymatic digestion treatment temperature is 20-50 ℃, including but not limited to 21 ℃, 22 ℃,25 ℃, 26 ℃, 28 ℃, 35 ℃, 40 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃ or 49 ℃, for 1-50 min, including but not limited to 2min, 3min, 4min, 5min, 6min, 7min, 8min, 20min, 25min, 30min, 40min, 42min, 43min, 45min, 46min, 47min, 48min or 49min.

In an eighth aspect, the present invention provides a method for FISH detection of paraffin-embedded tissue samples, the method comprising:

and (3) preprocessing the paraffin-embedded tissue sample by using the preprocessing method for FISH detection of the paraffin-embedded tissue sample in the seventh aspect, and performing FISH detection on the preprocessed sample.

The method has the advantages that the designed pretreatment liquid is used for pretreating the old FFPE sample, so that the FISH probe can be promoted to enter a cell nucleus, the tissue shedding is reduced, the effective hybridization is realized, the fluorescence background is reduced, the FISH signal observation is facilitated, the FISH detection can be carried out on the old FFPE sample, the method has wide application prospects, and the method comprises the steps of carrying out backtracking research on the old sample or carrying out research on the first FFPE sample of a relapsing patient and the like.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, a brand-new pretreatment liquid formula is developed, and all components are cooperatively matched, so that the FISH probe can be effectively promoted to enter a cell nucleus aiming at an old FFPE sample with the preparation time of more than 3 years, the tissue exfoliation is reduced, the fluorescence background is effectively hybridized, the FISH signal observation is facilitated, and the method can be effectively applied to the retrospective research of the old sample or the research of the first FFPE sample of a relapsed patient.

Drawings

FIG. 1 is a graph of a 120 Xmagnification contrast group of DAPI stained gastric tissues in example;

FIG. 2 is a drawing of an experimental group of 120 Xtimes microscopic DAPI-stained stomach tissues of example;

FIG. 3 is a view of a control group of mammary gland tissues under 1100 Xtimes microscope according to example;

FIG. 4 is a graph of an experimental group of mammary gland tissues under 1100 Xtimes microscope in example;

FIG. 5 is a graph of fluorescence signals of the experimental group of example 2, the signals are uniformly lighted and have clear edges;

FIG. 6 is a graph of fluorescence signals of the experimental group of example 3, the signals are uniformly lighted and have clear edges;

FIG. 7 is a graph of fluorescence signals of the experimental group of example 4, the signal points are bright and the edges are clear;

FIG. 8 is a graph of fluorescence signals of the experimental group of example 5, the signal points are bright and the edges are clear;

FIG. 9 is a graph of fluorescence signals of the experimental group of example 6, the signal points are bright and the edges are clear;

FIG. 10 is a graph of fluorescence signals of the experimental group of example 7, the signal points are bright and the edges are clear;

FIG. 11 is a photograph of a control group under a low power lens of example 8;

FIG. 12 is a view of the experimental group under the low power lens of example 8;

FIG. 13 is a view of a control group under high power lens of example 8;

FIG. 14 is a diagram of experimental groups under high power lens in example 8;

FIG. 15 is a graph of fluorescence signals of the experimental group of example 9, with bright signal spots and clear edges;

FIG. 16 is a diagram of the experimental group under the high power microscope of example 10, in which 80% or more of the nuclei are exposed clearly;

FIG. 17 is a diagram of the experimental group under high power lens of example 11, showing obvious swelling deformation of cells;

FIG. 18 is a diagram of the experimental group under high power lens of example 12;

FIG. 19 is a graph of fluorescent signals of comparative example 1, with a signal hybridization rate of about 50% in red and green channels;

FIG. 20 is a graph of fluorescence signals of comparative example 2, the hybridization rate of red and green channel signals is about 30%, and the signal spot size and brightness of cells hybridized successfully are weaker than those of the example;

FIG. 21 is a graph of fluorescent signals of comparative example 3, the hybridization rate of red and green channel signals is about 30%, and the signal spot size and brightness of cells hybridized successfully are weaker than those of the example;

FIG. 22 is a graph of fluorescent signals of comparative example 4, with a hybridization rate of red and green channel signals ≈ 10%.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available from the normal sources.

Example 1

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology Ltd., A610185) 15mM, naCl (Guangdong wide reagent technology Ltd., AR 500) 50mM, tris-HCl (Tris: biotechnology Ltd., shanghai) A600194; HCl: guangdong wide reagent technology Ltd., AR 500) 10mM, tritonX-100 (Shanghai Allantin Biotechnology Ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology Ltd., A600560) (mass%) 1%.

The preparation method of the pretreatment liquid comprises the following steps: tritonX-100 was stored at-20 ℃ for 30min, 2.9g of NaCl was weighed, 30mL of 0.5M EDTA solution and 10mL of 1M Tris-HCl (pH 8.0) were added, after NaCl was sufficiently dissolved, 5g of Trition X-100 and 10g of Twenn 20 were added, and magnetic stirring was carried out for 30min to reach a constant volume of 1L.

2 samples (thickness 3 μm) of neutral formalin-fixed paraffin-embedded breast tissue sections prepared in 2018 and 2 samples (thickness 3 μm) of neutral formalin-fixed paraffin-embedded stomach tissue sections prepared in 2017 were selected. Baking slices in hot bench for 20min, adding dewaxing agent 10min × 2 jar, soaking in 25 deg.C anhydrous ethanol for 10min, sequentially adding anhydrous ethanol, 90% ethanol, and 70% ethanol, respectively soaking for 3min, soaking in purified water for 3min, and removing excessive water.

Experimental groups: putting the treated slices into the pretreatment liquid at 95 ℃ and boiling for 25min; control group: and putting the processed slices into purified water at 99 ℃ for boiling for 25min, taking out the slides, and airing at 25 ℃. 200 μ L of protease (20160321 # Yuejiu) preheated at 37 deg.C for in situ hybridization is dropped into the sample area, and digested for 5-20 min. Throwing off the redundant liquid, putting into 2 XSSC at 25 ℃ for 3min, then sequentially putting into 70% ethanol, 90% ethanol and absolute ethanol, respectively soaking for 2min, taking out the slide, and airing at 25 ℃. Taking out HER2 gene detection kit (fluorescence in situ hybridization method) from a refrigerator at the temperature of-20 ℃ and carrying out national mechanical standard 20213400138, shaking, mixing uniformly, carrying out instantaneous centrifugation, adding 10 mu L of probes to a slide hybridization area, covering a cover glass with the thickness of 22 multiplied by 22mm, and lightly pressing to uniformly distribute the probes to avoid generating bubbles. The coverslip and slide contacting portions were completely covered with rubber cement along the coverslip edges. Putting the slide into a hybridization instrument, wetting a wet strip of the in-situ hybridization instrument, putting the wet strip into a wet strip clamping position, covering an upper cover of the hybridization instrument, setting a program of Denat & Hyb, and performing denaturation at 85 ℃ for 5min and hybridization at 37 ℃ for 18 hours. After hybridization, the rubber cement was torn off, the slide was removed, the slide was placed in 2 XSSC at 37 ℃ for 10min, then 0.1 NP40 (2 XSSC) for 5min, then placed in 70% ethanol at 25 ℃ for 3min, the slide was taken out, air dried in the dark, 10. Mu. LDAPI solution (blue staining solution for in situ hybridization (20160322 # for Yueji instruments)) was dropped onto the dried 22X 22mm slide, the sample piece was inverted, the slide was brought into contact with the target area of the slide, and air bubbles were removed by gentle pressure inversion.

Observing the whole tissue piece under a low power microscope, as shown in figures 1 and 2, dyeing stomach tissues under a 20X power microscope by DAPI, wherein over 80% of visual field nuclei of tissues of an experimental group are exposed and clear, and the number of cells is slightly more than that of cells of a control group; observing cells under a high power lens, wherein the DAPI staining of an experimental group is uniform and is clearly compared with the background, and the DAPI staining of a control group is weak and has a cavity feeling and is clearly compared with the background; HER2 (red) and CEP17 (green) signals are observed through a specific channel filter, as shown in figures 3 and 4, signal points of an experimental group are bright and have clear edges, the hybridization rate of red and green channel signals is 85%, red and green signal points of more than 60% of cells of a control group are fine, almost 30% of cells can not see signals, and non-specific red impurities are adsorbed on part of backgrounds. The detection effect of the pretreatment liquid is obviously better than that of the conventional treatment.

Example 2

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology Ltd., A610185) 10mM, naCl (Guangdong wide reagent technology Ltd., AR 500) 50mM, tris-HCl (Tris: biotechnology Ltd., shanghai) A600194; HCl: guangdong wide reagent technology Ltd., AR 500) 10mM, triton X-100 (Shanghai Aladdin Biotechnology Ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology Ltd., A600560) (mass%) 1%.

The preparation method of the pretreatment liquid refers to example 1.

Selecting 5 neutral formalin fixed paraffin embedded gastric tissue section samples (thickness 3 μm) prepared in 2017, heating, baking for 20min, adding dewaxing agent 10min × 2 jar, absolute ethanol at 25 deg.C for 10min, sequentially adding absolute ethanol, 90% ethanol, and 70% ethanol, soaking for 3min, adding purified water for 3min, and removing excessive water. Putting the glass slide into pretreatment liquid at 99 ℃ for boiling for 20min, taking out the glass slide for drying at 25 ℃, dropwise adding 200 mu L of protease (20160321 # in Guangdong ear) for in-situ hybridization preheated at 37 ℃ into a sample area, digesting for 5-20 min, throwing off redundant liquid, putting the glass slide into 2 XSSC at 25 ℃ for 3min, sequentially putting the glass slide into 70% ethanol, 90% ethanol and absolute ethanol for 2min respectively, taking out the glass slide for drying at 25 ℃ and drying, taking out a HER2 gene detection kit (fluorescence in-situ hybridization method) from a refrigerator at-20 ℃ for national instrument injection 20213400138, shaking, uniformly mixing, instantly centrifuging, adding 10 mu L of probes into a hybridization area, covering a cover glass with the size of 22X 22mm, and uniformly distributing the probes by light pressure to avoid generating bubbles. The coverslip and slide contacting portion were completely covered with rubber cement along the coverslip edge. Putting the slide into a hybridization instrument, wetting a wet strip of the in-situ hybridization instrument, putting the wet strip into a wet strip clamping position, covering an upper cover of the hybridization instrument, setting a program of 'Denat & Hyb', and hybridizing for 18 hours at the denaturation temperature of 85 ℃,5 minutes and 37 ℃. After hybridization, the rubber cement was peeled off, the cover glass was removed, the resultant was put in 2 XSSC at 37 ℃ for 10min, and then 0.1% NP40/2 XSSC for 5min, and the resultant was put in 70% ethanol at 25 ℃ for 3min, and the slide glass was taken out and dried in the dark. Drop 10 μ l of the api solution (in situ hybridization blue stain 20160322 (yue ear instrument)) onto a dry 22 × 22mm coverslip, invert the sample piece to bring the coverslip into contact with the target area of the slide, invert and gently press to remove air bubbles.

Observing the whole tissue slice under a low power microscope, wherein over 80 percent of visual field cell nuclei are naked and clear, and when observing cells under a high power microscope, DAPI staining is uniform and is clear in comparison with a background; the HER2 (red) and CEP17 (green) signals were observed through a specific channel filter, and the hybridization rate of the signals in the red and green channels was 84%, as shown in fig. 5, the signal spots were bright and uniform, and the edges were clear.

Example 3

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology, A610185) 50mM, naCl (Guangdong Wide reagent science, AR 500) 50mM, tris-HCl (Tris: biotechnology, shanghai) A600194; HCl: guangdong reagent science, AR 500) 10mM, triton X-100 (Shanghai Aladdin Biotechnology, inc., T109026) 0.5% by mass, and Tween20 (Biotechnology, shanghai) 1% by mass.

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

The whole tissue slice is observed under a low power microscope, and over 80 percent of visual field cell nucleuses are exposed and clear. Cells are observed under a high power lens, the DAPI staining is uniform, and the contrast with the background is clear; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 6, with bright, uniform signal spots and sharp edges.

Example 4

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology Ltd., A610185) 15mM, naCl (Guangdong wide reagent technology Ltd., AR 500) 150mM, tris-HCl (Tris: biotechnology Ltd., shanghai) A600194; HCl: guangdong wide reagent technology Ltd., AR 500) 10mM, triton X-100 (Shanghai Allantin Biotechnology Ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology Ltd., A600560) (mass%) 1%.

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

Observing the whole tissue slice under a low power microscope, wherein over 80 percent of visual field cell nucleuses are exposed and clear, and observing the cells under a high power microscope, the DAPI staining is uniform, and the contrast with the background is clear; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 7, with bright signal spots and sharp edges.

Example 5

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology Ltd., A610185) 15mM, naCl (Guangdong wide reagent technology Ltd., AR 500) 300mM, tris-HCl (Tris: biotechnology Ltd., shanghai) A600194; HCl: guangdong wide reagent technology Ltd., AR 500) 10mM, triton X-100 (Shanghai Allantin Biotechnology Ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology Ltd., A600560) (mass%) 1%.

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

The whole tissue slice is observed under a low power microscope, and over 80 percent of visual field cell nucleuses are exposed and clear. Cells are observed under a high power lens, the DAPI staining is uniform, and the contrast with the background is clear; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 8, with bright signal spots and sharp edges.

Example 6

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology industries, ltd., A610185) 15mM, naCl (Guangdong wide reagent science Co., ltd., AR 500) 50mM, tris-HCl (Tris: biotechnology industries, ltd., A600194; HCl: guangdong wide reagent science Co., ltd., AR 500) 5mM, triton X-100 (Shanghai Arlatin Biotechnology science Co., ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology industries, ltd., A600560) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded samples of stomach tissue sections (thickness 3 μm) prepared in 5 cases in 2017 were selected, and the pretreatment and FISH detection methods were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and over 80 percent of visual field cell nucleuses are exposed and clear. Cells are observed under a high power lens, the DAPI staining is uniform, and the contrast with the background is clear; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 9, with bright signal spots and sharp edges.

Example 7

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology industries, ltd., A610185) 15mM, naCl (Guangdong wide reagent science Co., ltd., AR 500) 50mM, tris-HCl (Tris: biotechnology industries, ltd., A600194; HCl: guangdong wide reagent science Co., ltd., AR 500) 50mM, triton X-100 (Shanghai Arlatin Biotechnology science Co., ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology industries, ltd., A600560) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded samples of stomach tissue sections (thickness 3 μm) prepared in 5 cases in 2017 were selected, and the pretreatment and FISH detection methods were performed according to example 2.

The whole tissue slice is observed under a low power microscope, and over 80 percent of visual field cell nucleuses are exposed and clear. Cells are observed under a high power lens, the DAPI staining is uniform, and the contrast with the background is clear; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 10, with bright signal spots and sharp edges.

Example 8

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology, A610185) 30mM, naCl (Guangdong wide reagent science, AR 500) 150mM, tris-HCl (Tris: biotechnology, shanghai) A600194; HCl: guangdong wide reagent science, AR 500) 25mM, PEG400 (Biotechnology, shanghai) A611781) 1% (mass%) and Tween20 (Biotechnology, shanghai) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded lung tissue section samples (3 μm thick) prepared in 5 cases in 2019 were selected, and the pretreatment and FISH detection methods were performed according to example 1. The detection kit is a ROS1 gene breakage detection kit (fluorescence in situ hybridization method).

The whole tissue piece is observed under a low power microscope, as shown in figures 11 and 12, over 80% of the cell nuclei in the experimental group are exposed clearly, and the cell number is slightly more than that of the control group. When cells are observed under a high power lens, DAPI staining of an experimental group and DAPI staining of a control group have certain mottle feeling, and the contrast with the background is clear; the signals of the red end of the ROS1 and the green end of the ROS1 are observed through a specific channel filter, as shown in figures 13 and 14, the signal points of the experimental group are sharp and bright and uniform, the signal hybridization rate is more than 80%, the signal hybridization rate of the control group in the red and green channel is less than 30%, the cells which are hybridized successfully have the signal points with the size equal to the brightness of the experimental group.

Example 9

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology, A610185) 100mM, naCl (Guangdong wide test agent science, AR 500) 150mM, tris-HCl (Tris: biotechnology, shanghai) A600194; HCl: guangdong wide test agent science, AR 500) 5mM, SDS (Biotechnology, shanghai) 0.15% (mass%) and tert-butanol (Biotechnology, shanghai) 10% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded mammary tissue section specimens (thickness 3 μm) prepared in 5 cases 2018 were selected, and the pretreatment and FISH detection methods were according to example 2.

The whole section is observed under a low power lens, the digestion of tissues is observed to be complete, over 80 percent of visual field cell nuclei are exposed and clear, DAPI staining is slightly mottled and is clearly compared with the background, then HER2 and CEP17 signals are observed under a 100-time high power lens through a specific channel optical filter, as shown in figure 15, signal points are bright, and the edges are clear.

Example 10

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and a method for FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology Ltd., A610185) 15mM, naCl (Guangdong wide reagent technology Ltd., AR 500) 600mM, tris-HCl (Tris: biotechnology Ltd., shanghai) A600194; HCl: guangdong wide reagent technology Ltd., AR 500) 10mM, triton X-100 (Shanghai Allandin Biotechnology Ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology Ltd., A600560) (mass%) 1%.

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and the cell nucleus with more than 80 percent of visual field is exposed and clear when the tissue is observed. When the cells were observed under high power microscope, as shown in fig. 16, the cells were significantly swollen and deformed, stained uniformly with DAPI, and clearly compared with the background. HER2 and CEP17 signals were observed through specific channel filters under a 100-fold high power microscope, with slightly weaker signal points.

Example 11

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and FISH detection using the same.

The pretreatment liquid contains: EDTA (Bio-engineering, A610185) 15mM, naCl (Guangdong wide reagent science and technology Co., ltd., AR 500) 50mM, tris-HCl (Tris: bio-engineering, shanghai) Co., ltd., A600194; HCl: guangdong wide reagent science and technology Co., AR 500) 100mM, triton X-100 (Shanghai Aradin Biochemical science and technology Co., ltd., T109026) 0.5% by mass and Tween20 (Bio-engineering, shanghai) Co., ltd., A600560) 1% by mass.

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and the cell nucleus with more than 80 percent of visual field is exposed and clear when the tissue is observed. When the cells were observed under high power microscope, as shown in fig. 17, the cells were significantly swollen and deformed, had more tissues that could not be digested, and the contrast effect was poor. HER2 and CEP17 signals were observed through specific channel filters under a 100-fold high power microscope, with slightly weaker signal points.

Comparing example 2 with examples 10 and 11, it is understood that the concentration of sodium chloride and buffer in the pretreatment liquid is controlled according to the present application, and the swelling and deformation of cells can be effectively prevented and the signal intensity can be improved.

Example 12

The present embodiment provides a pretreatment liquid for FISH detection of FFPE samples and FISH detection using the same.

The pretreatment liquid contains: EDTA (Biotechnology, shanghai, inc., A610185) 30mM, KCl (Guangdong wide reagent technology, AR 500) 150mM, tris-HCl (Tris: biotechnology, shanghai, inc., A600194; HCl: guangdong wide reagent technology, AR 500) 25mM, NP40 (Biotechnology, shanghai, A600385) 0.5% (mass%) and Tween20 (Biotechnology, shanghai, inc., A600560) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded lung tissue section samples (3 μm thick) prepared in 5 cases in 2019 were selected, and the pretreatment and FISH detection methods were performed according to example 1. The detection kit is a ROS1 gene breakage detection kit (fluorescence in situ hybridization method).

And observing the whole section under a low power lens, wherein the tissue digestion is completely observed, over 80 percent of visual field cell nuclei are exposed and clear, DAPI staining is slightly mottled, contrast with the background is clear, and signals of an ROS1 red end and an ROS1 green end are observed through a specific channel filter, as shown in figure 18, signal points are sharp and bright, and edges are clear.

Comparative example 1

The comparative example provides a pretreatment liquid for FISH detection of an FFPE sample and FISH detection by using the pretreatment liquid.

The pretreatment liquid contains: naCl (Guangdong wide reagent science Co., ltd., AR 500) 50mM, tris-HCl (Tris: bio-engineering (Shanghai) Co., ltd., HCl: guangdong wide reagent science Co., ltd., AR 500) 10mM, triton X-100 (Shanghai Azading Biotechnology Co., ltd., T109026) 0.5% (mass%) and Tween20 (Bio-engineering (Shanghai) Co., ltd., A600560) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and 50% of visual field cell nucleuses are exposed and clear when the tissue is observed. Cells are observed under a high power lens, the DAPI staining is uniform, and the contrast with the background is clear; HER2 (red) and CEP17 (green) signals are observed through a specific channel filter, as shown in FIG. 19, the hybridization rate of the signals in red and green channels is about 50%, which indicates that the pretreatment solution lacks EDTA, and the signal hybridization rate is obviously reduced.

Comparative example 2

The comparative example provides a pretreatment liquid for FISH detection of an FFPE sample and FISH detection by using the pretreatment liquid.

The pretreatment liquid contains: EDTA (Biotechnology, A610185) 15mM, tris-HCl (Tris: biotechnology, shanghai) GmbH A600194; HCl: guangdong reagent technology, inc., AR 500) 10mM, triton X-100 (Shanghai Aladdin Biotechnology, inc., T109026) 0.5% (mass%), and Tween20 (Biotechnology, shanghai) GmbH, A600560) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded samples of stomach tissue sections (thickness 3 μm) prepared in 5 cases in 2017 were selected, and the pretreatment and FISH detection methods were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and the cell nucleus with more than 50 percent of visual field is exposed and clear when the tissue is observed. Cells are observed under a high power lens, DAPI staining is uniform, and part of cells have cloud feeling; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 20, the hybridization rate of the red and green channel signals was about 30%, the signal spot size and brightness of the cells that hybridized successfully were weaker than those of the examples, indicating that the pretreatment solution lacks sodium chloride, resulting in a significant decrease in the signal hybridization rate and a decrease in the signal.

Comparative example 3

The comparative example provides a pretreatment liquid for FISH detection of an FFPE sample and FISH detection by using the pretreatment liquid.

The pretreatment liquid contains: EDTA (Biotechnology industries, ltd., A610185) 15mM, naCl (Guangdong wide reagent science and technology Co., ltd., AR 500) 50mM, triton X-100 (Shanghai Allantin Biotechnology science and technology Co., ltd., T109026) 0.5% (mass%) and Tween20 (Biotechnology industries, ltd., A600560) 1% (mass%).

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded samples of stomach tissue sections (thickness 3 μm) prepared in 5 cases in 2017 were selected, and the pretreatment and FISH detection methods were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and the cell nucleus with more than 50 percent of visual field is exposed and clear when the tissue is observed. Cells are observed under a high power lens, DAPI staining is uniform, and part of cells have cloud feeling; HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 21, the hybridization rate of the red and green channel signals was about 30%, the signal spot size and brightness of the cells that hybridized successfully were weaker than those of the examples, indicating that the pretreatment solution lacks buffer solution, resulting in a significant decrease in signal hybridization rate and a decrease in signal.

Comparative example 4

The comparative example provides a pretreatment liquid for FISH detection of an FFPE sample and FISH detection by using the pretreatment liquid.

The pretreatment liquid contains: EDTA (Biotechnology, inc., A610185) 15mM, naCl (Guangdong reagent science, inc., AR 500) 50mM, tris-HCl (Tris: biotechnology, inc., shanghai) A600194; HCl: guangdong reagent science, inc., AR 500) 10mM.

The preparation method of the pretreatment liquid refers to example 1.

2 neutral formalin-fixed paraffin-embedded stomach tissue section specimens (thickness 3 μm) prepared in 5 cases 2017 were selected, and pretreatment and FISH detection were performed according to example 2.

The whole tissue piece is observed under a low power microscope, and the cell nucleus with more than 50 percent of visual field is exposed and clear when the tissue is observed. Observing cells under a high power microscope, wherein DAPI staining is uniform, and connective tissue wrapping fog feeling exists on the outer layer of the cells; the HER2 (red) and CEP17 (green) signals were observed through specific channel filters, as shown in fig. 22, the hybridization rate of the red and green channel signals was ≈ 10%, and the signal spot size and brightness of the cells hybridized successfully were weaker than those of the examples, indicating that the pretreatment solution lacked the clearing agent component, resulting in a significant decrease in the signal hybridization rate and a reduction in the signal.

In conclusion, the invention develops a brand-new pretreatment liquid formula, EDTA is used as a crosslinking release buffer component, the alkaline metal chloride provides a cationic environment, the flaking is reduced, the crosslinking is released gently, the buffer provides a buffer environment, the fluctuation of the pH value in the process of boiling slices is reduced, the transparentizing agent dissolves partial lipid and components, the cells become more uniform and transparent, and the components are matched in a synergistic manner, so that the FISH probe can be promoted to enter the cell nucleus of an old sample which is prepared for more than 3 years, the tissue shedding is reduced, the effective hybridization is realized, the fluorescence background is reduced, and the FISH signal observation is facilitated.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (10)

1. A pretreatment liquid for FISH detection of paraffin-embedded tissue samples is characterized by comprising a protective liquid and a transparent agent;

the protective solution contains ethylenediamine tetraacetic acid, alkali metal chloride and buffer solution;

the clearing agent contains any one or the combination of at least two of tertiary butanol, polyethylene glycol or surfactant.

2. The pretreatment fluid for FISH testing of a paraffin-embedded tissue specimen according to claim 1, wherein the basic metal chloride comprises any one or a combination of at least two of sodium chloride, potassium chloride or lithium chloride;

preferably, the buffer solution comprises any one of a Tris-HCl buffer solution, a potassium dihydrogen phosphate-sodium hydroxide buffer solution, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution or a barbituric sodium-hydrochloric acid buffer solution;

preferably, the average relative molecular mass of the polyethylene glycol is 200 to 1500;

preferably, the surfactant comprises any one or a combination of at least two of polyethylene glycol p-isooctyl phenyl ether, dodecyl sulfate, tween or NP 40;

preferably, the polyethylene glycol p-isooctyl phenyl ether comprises polyethylene glycol p-isooctyl phenyl ether-100, polyethylene glycol p-isooctyl phenyl ether-114;

preferably, the lauryl sulfate salt comprises sodium lauryl sulfate and/or lithium lauryl sulfate;

preferably, the tween comprises tween20 and/or tween 80.

3. The pretreatment liquid for FISH detection of a paraffin-embedded tissue sample according to claim 1 or 2, wherein the concentration of ethylenediamine tetraacetic acid in the pretreatment liquid is 10 to 100mmol/L;

preferably, the concentration of the alkali metal chloride in the pretreatment liquid is 50 to 300mmol/L;

preferably, the concentration of the buffer salt in the pretreatment liquid is 5 to 50mmol/L;

preferably, the mass percent of the clearing agent in the pretreatment liquid is 0.5-10%.

4. Use of the pretreatment fluid for FISH examination of a paraffin-embedded tissue sample according to any one of claims 1 to 3 for the preparation of a product for examination of a paraffin-embedded tissue sample.

5. A paraffin-embedded tissue sample pretreatment kit, comprising the pretreatment solution for FISH assay of paraffin-embedded tissue samples according to any one of claims 1 to 3.

6. A paraffin embedded tissue sample FISH assay kit, comprising the pretreatment liquid for paraffin embedded tissue sample FISH assay of any one of claims 1-3;

preferably, the kit further comprises FISH detection reagents.

7. Use of the pretreatment liquid for FISH detection of paraffin-embedded tissue samples according to any one of claims 1 to 3 in FISH detection of paraffin-embedded tissue samples.

8. A pretreatment method for FISH detection of paraffin-embedded tissue samples, which is characterized by comprising the following steps:

sequentially carrying out slicing treatment, baking treatment and dewaxing treatment on a paraffin-embedded tissue sample to be detected, mixing the dewaxed sample with the pretreatment liquid for FISH detection of the paraffin-embedded tissue sample in any one of claims 1-3, carrying out heating treatment, and carrying out enzyme digestion treatment on the heated sample.

9. The pre-treatment method for FISH detection of a paraffin embedded tissue sample according to claim 8, wherein the temperature of the heating treatment is 60 to 95 ℃ for 10 to 2000min;

preferably, the tool enzyme for the enzymatic digestion treatment comprises any one or a combination of at least two of proteinase K, pepsin or trypsin;

preferably, the enzyme digestion treatment temperature is 20-50 ℃, and the time is 1-50 min.

10. A method for FISH detection of paraffin-embedded tissue samples, the method comprising:

the method of claim 8 or 9, wherein the paraffin-embedded tissue sample is pre-treated and the pre-treated sample is subjected to FISH detection.

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