CN103382499B - Specimen pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization method and reagent kit thereof - Google Patents
Specimen pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization method and reagent kit thereof Download PDFInfo
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Abstract
A specimen pretreatment method for reducing autofluorescence interference in a fluorescence in situ hybridization method, comprising: the method comprises the following steps: formalin-fixed paraffin-embedded tissues; step two: slicing and baking; step three: sodium thiocyanate action; step four: adding 20 μ l of type IV collagenase with concentration of 5-10mg/mL or elastase with concentration of 1mg/mL into tissue with area of 24 × 32mm, sealing, and acting at 37 deg.C for 0.5-1hr to degrade extracellular matrix with autofluorescence property; step five: pepsin was used at 37 ℃. The invention also provides a reagent kit for the pre-treatment method of the specimen, which can be used for reducing the autofluorescence interference in the fluorescence in situ hybridization method.
Description
Technical field
The invention provides a kind of corpse or other object for laboratory examination and chemical testing pre-treating process for reducing fluorescent interference autologous in Fluorescent in situ hybridization, espespecially it technically provides a kind of at Fluorescent in situ hybridization (FISH, Fluorescence In Situ Hybridization) corpse or other object for laboratory examination and chemical testing pre-treatment step increase the 4th collagen type enzyme (collagenase type IV) or elastoser (elastase) treatment step, in order to reduce the autologous fluorescence of human tissue, improve the resolution of Fluorescent in situ hybridization fluorescent probes signal.
Background technology
Fluorescent in situ hybridization (FISH) is the technology that clinical diagnosis often uses, and can be used for early diagnosis, the selection of therapeutic modality or the activity of monitoring of diseases.As: patients with mastocarcinoma uses Herceptin (trastuzumab) treatment to be detect human epidermal growth factor acceptor-2 (HER2) gene expression according to Fluorescence in situ hybridization (FISH) or use when immunohistochemical staining (IHC) presents human epidermal growth factor acceptor-22+ (HER22+) gene Fluorescence in situ hybridization (FISH) to do further confirmation again, filters out the more medicable patient of medicine, in addition, Nucleophosmin-anaplastic lymphoma kinase and the two target spot micromolecular inhibitor of hepatocyte growth factor receptor (ALK/C-met) is accepted at echinoderms microtubule bindin 4 and non-small cell adenocarcinoma of lung (NSCLC) patient of Nucleophosmin-anaplastic lymphoma kinase (EML4-ALK) the fusion gene positive, the tumor regression of the patient Ke Da more than 90% that Ke Zhuo treats for Buddhist nun (Crizotinib), the patient of 57% obtains Objective responses, therefore, effectively detect that echinoderms microtubule bindin 4 and Nucleophosmin-anaplastic lymphoma kinase (EML4-ALK) fusion gene are very helpful and benefit for clinical target therapy.
Fluorescent in situ hybridization is under the principle not destroying deoxyribose nucleic acid chain-like structure, by double-strand deoxyribose nucleic acid chains by sex change original position separately, the deoxyribose nucleic acid probe demarcated with fluorescent and strand deoxyribose nucleic acid chains are carried out hybridization and are combined, finally under luminescence microscope, directly observe Fluorescent signal, in analysis of cells karyomit(e), whether have this sequence corresponding to deoxyribose nucleic acid probe.At present, the conventional fluorescent substance demarcating deoxyribose nucleic acid or antibody is FITC Fluorescein Isothiocyanate, green (the Oregon green) 488 in Ao Le hilllock, spectrum green (SpectrumGreen), because tissue has endogenous fluorescent group (endogenous fluorophores) as collagen protein, elastin, nicotinamide adenine dinucleotide reduced NADH, vitamin B2 phosphate (FMN, Flavin mononucleotide), flavin adenine dinucleotide (FAD, Flavin adenine dinucleotide) and die aromatischen Aminosaeuren (aromatic amino acids), if a corpse or other object for laboratory examination and chemical testing directly can affect the accuracy of Fluorescence in situ hybridization art and immunofluorescence dyeing interpretation with the autologous fluorescent of strong background, this type of endogenous material also similar short-wavelength light of Absorbable rod and produce exciting light, cause this type of autologous fluorescence institute reasons for its use light can reduce the ratio of fluorescent probes or antibody signal and background fluorescent, thus cause the resolution of fluorescent signal and the bad of quality, signal counting and interpretation can be affected.
Summary of the invention
Previous Fluorescent in situ hybridization corpse or other object for laboratory examination and chemical testing pre-treatment step is the victory peptide bond interrupting protein with stomach en-, but stomach en-deficiency improves the phenomenon of the autologous fluorescent of a corpse or other object for laboratory examination and chemical testing.
For improving the problems referred to above, the invention provides a kind of corpse or other object for laboratory examination and chemical testing pre-treating process for reducing fluorescent interference autologous in Fluorescent in situ hybridization, comprising the following steps:
Step one: paraffin embedding (FFPE) tissue fixed by formalin;
Step 2: section and baking sheet;
Step 3: Sodium Thiocyanate 99 (NaSCN) acts on;
Step 4: add 20 μ l concentration 5-10mg/mL the 4th collagen type enzyme or 1mg/mL elastoser at the tissue of area 24 × 32mm, in 37 DEG C of effect 0.5-1hr after mounting, the extracellular matrix of the autologous fluorescent property of tool of degrading; And
Step 5: with stomach en-(pepsin) 37 DEG C of effects.
The present invention separately provides two kinds for reducing the reagent cover group of the corpse or other object for laboratory examination and chemical testing pre-treating process of fluorescent interference autologous in Fluorescent in situ hybridization again, the 4th collagen type enzyme (the collagenase type IV) 100mg that the clostridium histolyticum (clostridium histolyticum) that one takes from purity 100% produces is dissolved in the phosphoric acid buffer (PBS of 10mL, phosphate buffered saline) after, store at the temperature of-20 DEG C with concentration 10mg/mL; Another gets the first type pancreatic elastase (the ELASTASE PANCREATIC TYPE I) 4mg of Mei Nong Du≤4.0units/mg in pig pancreas (PORCINE PANCREAS), be dissolved in 1mL phosphoric acid buffer (PBS, phosphate buffered saline) after, store at the temperature of-20 DEG C with concentration 4mg/mL.
The present invention increases the 4th collagen type enzyme or elastoser treatment step at the corpse or other object for laboratory examination and chemical testing pre-treatment step of Fluorescent in situ hybridization (FISH), degrade and there is the extracellular matrix of endogenous fluorescent group, significantly can reduce the autologous fluorescence of human tissue, improve fluorescent probes signal resolution degree, promote inspection quality and the exactness of Fluorescent in situ hybridization.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1: flow chart of steps of the present invention.
Fig. 2 (A): shown the corpse or other object for laboratory examination and chemical testing blank photo not using the inventive method process by luminescence microscope, the as seen strong autologous fluorescent of its tool.
Fig. 2 (B): use the corpse or other object for laboratory examination and chemical testing blank photo after the inventive method process by luminescence microscope display, its autologous fluorescent declines as seen.
Fig. 3 (A): by the autologous fluorescent sample of luminescence microscope display tool after stomach en-pre-treatment, carry out the photo of the Fluorescent in situ hybridization of HER2/CEP17, visible background fluorescent covers FISH probe signal.
Fig. 3 (B): after using the inventive method process by the autologous fluorescent sample of luminescence microscope display tool, carry out the photo of the Fluorescent in situ hybridization of HER2/CEP17, the autologous fluorescent of a visible corpse or other object for laboratory examination and chemical testing declines.
Fig. 4 (A): by the autologous fluorescent sample of luminescence microscope display tool after stomach en-pre-treatment, carry out the photo after the Fluorescent in situ hybridization background correction of HER2/CEP17, visual probe signal is unclear, cannot count.
Fig. 4 (B): after using the inventive method process by the autologous fluorescent sample of luminescence microscope display tool, carry out the photo after the Fluorescent in situ hybridization background correction of HER2/CEP17, the clear count enable of visual probe signal.
Fig. 5 (A): shown another corpse or other object for laboratory examination and chemical testing blank photo not using the inventive method process by luminescence microscope, as seen the strong autologous fluorescent of its tool.
Fig. 5 (B): use another corpse or other object for laboratory examination and chemical testing blank photo after the inventive method process by luminescence microscope display, its autologous fluorescent declines as seen.
Fig. 6 (A): by the autologous fluorescent sample of luminescence microscope display tool after stomach en-pre-treatment, carry out the photo after the Fluorescent in situ hybridization background correction of EML4/ALK, visual probe signal is unclear, cannot count.
Fig. 6 (B): after using the inventive method process by the autologous fluorescent sample of luminescence microscope display tool, carry out the photo after the Fluorescent in situ hybridization background correction of EML4/ALK, the clear count enable of visual probe signal.
Embodiment
Embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
In following embodiment, method therefor is ordinary method if no special instructions.
Consult shown in Fig. 1, the invention provides a kind of corpse or other object for laboratory examination and chemical testing pre-treating process for reducing fluorescent interference autologous in Fluorescent in situ hybridization, comprise following steps:
Step one (S1): paraffin embedding (FFPE) tissue fixed by formalin;
Step 2 (S2): section and baking sheet;
Step 3 (S3): Sodium Thiocyanate 99 (NaSCN) acts on;
Step 4 (S4): add 20 μ l concentration 5-10mg/mL the 4th collagen type enzyme or 1mg/mL elastoser at area 24 × 32mm on the tissue slice of above-mentioned process, in 37 DEG C of effect 0.5-1hr after mounting, degrade and there is the extracellular matrix of autologous fluorescent property; And
Step 5 (S5): with stomach en-(pepsin) 37 DEG C of effects.
In the pre-treating process of an above-mentioned corpse or other object for laboratory examination and chemical testing, in described step one (S1), describedly be organized as breast cancer, cancerous lung tissue, skin histology, the various tissue such as renal tissue, the concrete grammar fixing paraffin-embedded tissue with formalin is: being organized in mass concentration is soak in 10% neutral formalin damping fluid (10%neutral buffered formalin) to fix, set time is minimum is 6 hours, be no more than at most 48 hours, (first use after ethanol concn 95wt% reacts 3 hours through dehydration of alcohol again, re-use ethanol concn 99.8wt% and react 3 hours) after, soak dimethylbenzene (Xylene) again and make its transparence in 2 hours, paraffin (Paraffin) is finally used to embed (about 3 hours time).
In described step 2 (S2), the concrete grammar of section is: tool rest wax stone being placed in histotome clamps wax stone, 4 μm are cut in cold water with histotome, fish for section with slide to move in 50 DEG C of water-baths tissue slice is shakeout, again section being picked up with slide after tissue slice shakeouts makes slice sticker invest slide, uprightly made by slide the moisture of section remnants take advantage of a situation again to flow down, it firmly throws away by the moisture hand of residue; The concrete grammar drying sheet section is placed in 56 DEG C, baking oven, is dried to few more than 2 hours or overnight.
In described step 3 (S3), be by baking after section put into Sodium Thiocyanate 99 (NaSCN) solution that concentration is 1M, 80 DEG C heating 15-25min.Can assist to untie the fixing deoxyribose nucleic acid (DNA) that caused of formalin or protein is wound around alternately by the object that Sodium Thiocyanate 99 (NaSCN) processes.
In described step 5 (S5), the mass concentration of stomach en-(pepsin) is 0.005%, is joined on tissue slice after step 4, places the time of 3-10 minute at 37 DEG C.Be used to by the object that stomach en-(pepsin) processes the victory peptide bond interrupting protein, degrade proteins, reduce intact proteins produce the phenomenon of autologous fluorescent, and fluorescent probes is more easily combined with karyomit(e).
The present invention is with the 4th collagen type enzyme of suitable proportion (collagenase type IV) and the various wax stone tissue of elastoser (elastase) pre-treatment, can degrade and there is the extracellular matrix of endogenous fluorescent group, significantly can reduce the autologous fluorescence of human tissue, reduce the background signal of fluorescent detection type, improve fluorescent probes signal resolution degree, promote Fluorescent in situ hybridization inspection quality and exactness.Refer to shown in Fig. 2 (A), Fig. 2 (A) is observed with Fluorescein Isothiocyanate filter (spectra green FITC filter) by luminescence microscope, the brilliant white limit that the edge of the corpse or other object for laboratory examination and chemical testing processed without the 4th collagen type enzyme (collagenase type IV) or elastoser (elastase) presents is the part of fluorescent, the strong autologous fluorescent of display corpse or other object for laboratory examination and chemical testing blank tool; After corpse or other object for laboratory examination and chemical testing pretreatment process of the present invention process, present grey by the part of Fig. 2 (B) visible corpse or other object for laboratory examination and chemical testing edge fluorescent, represent that the autologous fluorescent of corpse or other object for laboratory examination and chemical testing blank declines.Fig. 3 (A) shows the autologous fluorescent sample of tool without the 4th collagen type enzyme (collagenase type IV) or elastoser (elastase) process merely through after stomach en-(pepsin) pre-treatment, carry out the Fluorescent in situ hybridization in human epidermal growth factor acceptor-2 and karyomit(e) 17 kinetochore (HER2/CEP17), show under luminescence microscope, original signal figure tool strong background, the ash group in figure represents that background fluorescent has covered Fluorescent in situ hybridization probe signal; And Fig. 3 (B) display is after corpse or other object for laboratory examination and chemical testing pretreatment process of the present invention process, the autologous fluorescent of a corpse or other object for laboratory examination and chemical testing declines, ash group in figure obviously reduces, represent that background fluorescent significantly reduces, white point in figure is high-visible, can provide and calculate signal and count, represent Fluorescent in situ hybridization probe signal resolution and count enable signal and count and all have remarkable increase.Separately refer to Fig. 4 (A), have autologous fluorescent sample without the 4th collagen type enzyme (collagenase type IV) or elastoser (elastase) process and merely through stomach en-(pepsin) pre-treatment after, carry out human epidermal growth factor acceptor-2 and karyomit(e) 17 kinetochore (HER2/CEP17) Fluorescent in situ hybridization, under luminescence microscope is observed, after background correction, probe signal is unclear, cannot count; And Fig. 4 (B) display is after corpse or other object for laboratory examination and chemical testing pretreatment process of the present invention process, the autologous fluorescent of a corpse or other object for laboratory examination and chemical testing declines, background fluorescent significantly reduces, Fluorescent in situ hybridization probe signal resolution and count enable signal are counted and are all had remarkable increase, white point in figure is high-visible, can provide and calculate signal and count, represent the clear count enable of probe signal after background correction.Refer to shown in Fig. 5 (A), by observing in luminescence microscope with Fluorescein Isothiocyanate filter (FITC filter), the brilliant white limit that the edge of the corpse or other object for laboratory examination and chemical testing processed without the 4th collagen type enzyme (collagenase type IV) or elastoser (elastase) presents is the part of fluorescent, and corpse or other object for laboratory examination and chemical testing blank has strong autologous fluorescent; After corpse or other object for laboratory examination and chemical testing pretreatment process of the present invention process, present grey by the part of the fluorescent at the visible corpse or other object for laboratory examination and chemical testing edge of Fig. 5 (B), represent that the autologous fluorescent of display corpse or other object for laboratory examination and chemical testing blank declines.Again Fig. 6 (A) represent have autologous fluorescent sample without the 4th collagen type enzyme (collagenase type IV) or elastoser (elastase) process and only after stomach en-(pepsin) pre-treatment, carry out echinoderms microtubule bindin 4 and Nucleophosmin-anaplastic lymphoma kinase (EML4-ALK) Fluorescent in situ hybridization, observe in luminescence microscope, ash group in original signal figure tool strong background figure represents that background fluorescent has covered Fluorescent in situ hybridization probe signal, after background correction, probe signal is unclear, cannot count; Fig. 6 (B) represents by after corpse or other object for laboratory examination and chemical testing pretreatment process of the present invention process, the autologous fluorescent of a corpse or other object for laboratory examination and chemical testing declines, ash group in figure obviously reduces, represent that background fluorescent significantly reduces, white point in figure is high-visible, calculating signal can be provided to count, represent background fluorescent and significantly reduce, Fluorescent in situ hybridization probe signal resolution and count enable signal are counted and are all had remarkable increase, the clear count enable of probe signal after background correction.
The present invention also provides two kinds of reagent cover groups of the corpse or other object for laboratory examination and chemical testing pretreatment process that can be used for autologous fluorescent interference in above-mentioned reduction Fluorescent in situ hybridization again, one gets the phosphoric acid buffer (PBS that the 4th collagen type enzyme (the collagenase type IV) 100mg produced by the clostridium histolyticum of purity 100% (clostridium histolyticum) is dissolved in 10mL, phosphate buffered saline) after, store at the temperature of-20 DEG C with concentration 10mg/mL; The first type pancreatic elastase (the ELASTASE PANCREATIC TYPE I) 4mg of Mei Nong Du≤4.0units/mg in pig pancreas (PORCINE PANCREAS) gets in another reagent system, be dissolved in 1mL phosphoric acid buffer (PBS, phosphatebuffered saline) after, store at the temperature of-20 DEG C with concentration 4mg/mL.This kind of reagent is used for restriction and shortcoming that corpse or other object for laboratory examination and chemical testing pre-treatment also can improve current Fluorescent in situ hybridization testing reagent cover group, reach effect identical with above-mentioned pre-treating process, therefore the present invention can be applicable to any corpse or other object for laboratory examination and chemical testing kind with autologous fluorescence (as corpse or other object for laboratory examination and chemical testing such as breast cancer, lung cancer, skin, kidneys).
Reagent of the present invention can allocate suitable concentration and action time according to different corpse or other object for laboratory examination and chemical testing, such as, when being used in the pre-treatment of a mammary tissue corpse or other object for laboratory examination and chemical testing, by the 4th above-mentioned collagen type enzyme reagent activity 5-10mg/mL, operative temperature 37 DEG C, and can act under 30-60 minute; Or when being used in the pre-treatment of a lung tissue corpse or other object for laboratory examination and chemical testing, by above-mentioned elastoser reagent activity 1mg/mL, operative temperature 37 DEG C, and can act under 30-60 minute.
In sum, the present invention can improve in existing Fluorescent in situ hybridization and carry out with stomach en-the problem that corpse or other object for laboratory examination and chemical testing pre-treatment step causes reducing the autologous fluorescence of a corpse or other object for laboratory examination and chemical testing, according to parallel laboratory test result verification, there is a corpse or other object for laboratory examination and chemical testing for strong autologous fluorescent, after the inventive method process, the autologous fluorescent of a corpse or other object for laboratory examination and chemical testing significantly declines to a great extent, relatively pepsin method, after process of the present invention, corpse or other object for laboratory examination and chemical testing probe signal can know counting, and probe signal resolution significantly significantly promotes.
Claims (3)
1., for reducing a corpse or other object for laboratory examination and chemical testing pre-treating process for fluorescent interference autologous in Fluorescent in situ hybridization, comprise following steps:
Step one: paraffin embedding (FFPE) tissue fixed by formalin; Describedly be organized as breast carcinoma tissue, cancerous lung tissue, skin histology or renal tissue;
Step 2: section and baking sheet;
Step 3: Sodium Thiocyanate 99 (NaSCN) acts on;
Step 4: add 20 μ l concentration 5-10mg/mL the 4th collagen type enzyme solution or 1mg/mL first type pancreatic elastase solution at the tissue of area 24 × 32mm, in 37 DEG C of effect 0.5-1hr after mounting, degrade and there is the extracellular matrix of autologous fluorescent property; And
Step 5: with stomach en-(pepsin) 37 DEG C of effects.
2. method as claimed in claim 1, it is characterized in that, the compound method of the 4th collagen type enzyme solution for: the 4th collagen type enzyme (the collagenase type IV) 100mg produced by the clostridium histolyticum (clostridium histolyticum) of purity 100% is dissolved in the phosphoric acid buffer (PBS of 10mL, phosphate bufferedsaline) after, store at the temperature of-20 DEG C with concentration 10mg/mL.
3. method as claimed in claim 1, it is characterized in that, the compound method of the first type pancreatic elastase solution is: the first type pancreatic elastase (the ELASTASE PANCREATIC TYPE I) 4mg getting Mei Nong Du≤4.0units/mg in pig pancreas (PORCINE PANCREAS), be dissolved in 1mL phosphoric acid buffer (PBS, phosphate bufferedsaline) after, store at the temperature of-20 DEG C with concentration 4mg/mL.
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| CN107478625A (en) * | 2017-08-04 | 2017-12-15 | 复旦大学附属中山医院 | Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method |
| CN108517366A (en) * | 2018-04-03 | 2018-09-11 | 四川大学 | Coriolis clostridium specific probe and application thereof |
| CN110208056B (en) * | 2019-06-06 | 2021-08-31 | 厦门大学附属第一医院 | Method for manufacturing tissue chip of gastric cancer HER-2 FISH |
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| CN101805728A (en) * | 2010-04-01 | 2010-08-18 | 北京金波绿泰科技有限公司 | Simple preparation method of activated pancreatic enzyme |
| CN102282252A (en) * | 2008-11-19 | 2011-12-14 | 人类起源公司 | Amnion derived adherent cells |
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| CN101805728A (en) * | 2010-04-01 | 2010-08-18 | 北京金波绿泰科技有限公司 | Simple preparation method of activated pancreatic enzyme |
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