CN106198174B - A kind of preprocess method of the histotomy for conventional FISH detection failure - Google Patents
A kind of preprocess method of the histotomy for conventional FISH detection failure Download PDFInfo
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Abstract
The present invention relates to a kind of preprocess methods of histotomy for conventional FISH detection failure.Specifically, the sodium citrate salt buffer of four kinds of various concentrations and pH value, the method for the histotomy to fail using the processing routine FISH detection of above-mentioned sodium citrate salt buffer and the kit comprising above-mentioned sodium citrate salt buffer are provided.Testing confirms to pre-process using the histotomy of sodium citrate salt buffer of the invention to conventional FISH detection failure, can detect that the example of 71%-73%, has detection success rate height, cheap advantage, be suitable for popularity application.
Description
Technical field
The present invention relates to biological detections and molecular diagnostic techniques field, specifically, being related to a kind of for routine FISH inspection
The preprocess method for the histotomy that dendrometry loses.
Background technique
FISH is the abbreviation of the English fluorescence in situ hybridization of fluorescence in situ hybridization, is referred to
One kind point of qualitative, positioning and relative quantification research is carried out to nucleic acid on histotomy, cell smear and chromosome sectioning etc.
Sub- biological method has many advantages, such as sensitive, special, intuitive.Under the conditions of its principle is " quenching " after high-temperature denaturation, fluorescence
The nucleic acid probe sequence of element label can be specifically bound in the target nucleic acid sequence of the homologous complementary in cell in-situ and target cell.
Common PCR and Nucleic acid sequencing techniques belong to nucleic acid molecules pathological diagnosis scope in the technology and clinical examination, but it is mesh
It is preceding to be uniquely able to maintain the intuitive gene diagnosis technology of in situ tissue cytomorphology structure, it can be observed that interphase cell and
The large area such as the change of each cell chromosome number, the amplification of gene local segment, missing and the sequence reorganization of division cycle
Gene mutation.The technology as one of molecular biology and the common technique of molecular pathology, be widely used in oncobiology,
The fields such as Hematopathology, heredity, microbiology, Cytobiology and molecular biology, Neuroendocrinology, immunology, especially exist
In treatment assessment and the individualized treatments medical regimens such as Index for diagnosis, FISH has been widely used in examining for tumour and other diseases
The disconnected, selection of therapeutic scheme, course for the treatment of monitoring and Index for diagnosis etc..
The simple process figure of FISH detection is as shown in Figure 1.Several sample treatments when FISH is detected mainly comprise the following steps,
After dimethylbenzene carries out dewaxing treatment and alcohol elution to slice, slice passes through sodium citrate salt buffer or sodium sulfocynanate
(Sodium Thiocyanate, NaSCN) processing exposes the specificity portion of FISH probe, then in conjunction with pepsin digestion
Carry out the hybridization of probe and the elution of non-specific binding.Therefore FISH detects whether effective it is crucial that guaranteeing probe matter
Under the premise of amount, two pre-treatment steps before FISH probe hybridization (cut by the heat treatment tissue such as sodium citrate salt buffer
Piece and pepsin digestion histotomy) in, it was not only able to maintain the integrality of histocyte morphosis, but also can be effectively removed
Gene probe is influenced to enter and be integrated to the obstacle in target gene site.
In the technology path of FISH detection, for being sliced the pretreated first step, domestic laboratory generallys use following
Two kinds of processing methods:
1) citrate buffer heats method: citrate buffer (Citrate Buffer, 10mM, pH6.0)
Perhaps hyperbaric heating is handled to progress enzymic digestion (pepsin or Proteinase K) after 80-100 DEG C common heating, removes big portion
Divide and FISH probe is hindered to enter protein of the nucleus in conjunction with nucleic acid target area.The maximum benefit of this method be it is cheap,
Operation is easy, reagent is nontoxic.Access have been reported that in clinical practice, sodiocitrate buffer concentration used
It is unified all to use 10mM, pH 6.0.
2) sodium sulfocynanate facture: it is reported that, compared with citrate buffer heating treatment method, sodium sulfocynanate
Facture flake chance increased significantly, in the slice for occurring especially in micro-assembly robot matrix (Tissue Microarray), while sulphur
Cymag has certain chemical toxicity, according to interaction encyclopaedia net introduction, belongs to the product of high environmental risk: easy to damp in air
Solution meets acid and generates toxic gas, and thyroid microcancer occurs in slow poisoning, maximum permissible concentration 50mg/m in air3, and thiocyanation
Sodium price is relatively expensive.
Unanimously think that the histotomy for being applied to FISH detection is fixed in 10% neutral formalin fixer at present
Specimens paraffin embedding slices.Using 10% neutral formalin fixer fixing organization, conventional dehydration, paraffin embedding are clinical common
And be presently the most the effective method for saving institutional framework, and the DNA information on chromosome can be saved well.
Due to the particularity of FISH detection, probe needs effectively through cytoplasm, cytoplasm and endonuclear each
Kind institutional framework reaches target gene position and combines with target gene specific, therefore it is required that: 1) tissue block is generally in 10% neutrality
Formalin thoroughly fixes 24-48 hours (no longer than 72 hours), by conventional dehydration and paraffin embedding step, preparation
At paraffin mass；2) tissue block of paraffin embedding is preferably (storage life is within 8 years) than comparatively fresh, in fixed tissue between
Crosslinking be not excessively oxidated；3) it is applied to the paraffin section of FISH detection with a thickness of 4-5 microns.
However, as FISH detection technique is increasingly being applied in clinical laboratory inspection, it is some to cause clinic
The case of FISH difficult diagnosis is consequently increased, such as weak FISH marking signal or nothing or cell within a cell interstitial is caused to carry on the back
Jing Taiqiang has covered FISH signal itself.The case of such difficult diagnosis includes:
1) postoperative tissue block is more than 72 hours in festivals or holidays or other set times in special circumstances；
2) it for the patient of tumor recurrence, needs to trace and compared with tissue block after primary tumo(u)r or original operation
Compared with the tissue block for being likely encountered paraffin embedding is stored more than 8 years even for more time；
3) for clinical medical retrospective study, usually to certain tumours more certain genetic fragments of FISH method
Mutation, judges influence of the gene for clinical diagnosis, treatment and prognosis；
4) cytoplasm of some tissues is finer and close, such as lung squamous cancer and certain sarcomas；
5) for some tissues, although FISH marking signal is still weaker after the factor of improvement other influences signal.
In clinical diagnosis, when there is the above problem, changes the protease digestion time by additional 1-2 trial and change
After becoming the conditions such as eluotropic strength, if signal still reaches to technically make diagnosis " failure " less than diagnostic techniques requirement
Conclusion belongs to acceptable scope, but produces obstruction to clinical diagnosis and related science research.Therefore it needs a kind of for miscellaneous
The processing method of the FISH slice of hardly possible beset by troubles.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide a kind of tissue for conventional FISH detection failure
The reagent treatment and method of slice.
In a first aspect, the present invention provides a kind of pretreating reagent of histotomy for conventional FISH detection failure,
The pretreating reagent is sodium citrate salt buffer, concentration 30-80mM, pH 6.2-7.1.
As a preference of the invention, the concentration and pH of the sodium citrate salt buffer are respectively selected from following each
Any group in group:
A) 30mM, pH6.2；
B) 40mM, pH6.5；
C) 60mM, pH6.8；
D) 80mM, pH7.1.
As another preference of the invention, the histotomy of the conventional FISH detection failure is 10% neutrality Fu Er
The fixed paraffin-embedded tissue slice of Malin's fixer, and be to eliminate protease digestion time, slice elution time and spy
Failure is detected under the premise of needle qualitative factor.
Second aspect, the present invention provides application of the pretreating reagent in FISH detection.
As a preference of the invention, the application is to be detected using the pretreating reagent to conventional FISH
The histotomy of failure carries out microwave heating pressurized treatments.
The third aspect, the present invention provides it is a kind of for conventional FISH detection failure histotomy preprocess method,
The method includes carrying out microwave using histotomy of the pretreating reagent to routine FISH detection failure to add
The step of hot pressurized treatments.
As a preference of the invention, the preprocess method specifically: detect the routine FISH and fail
Histotomy be placed in the dedicated pressure cooker of micro-wave oven, the pretreating reagent is added and is allowed to flood tissue completely and cuts
Piece upper limb, is heated in micro-wave oven with 1000-2000W, to hear stopping heating immediately after the faint outlet sound of heating air valve,
After maintaining high-pressure cooker cover to close 25-30 minutes under non-heated state, pot cover is opened, at room temperature natural cooling.
As another preference of the invention, the histotomy of the routine FISH detection failure is 10% neutral formal
72 hours paraffin-embedded tissue slices are fixed for more than in woods fixer, and when excluding protease digestion time, slice elution
Between and probe mass factor under the premise of detect failure；The pretreating reagent is sodium citrate salt buffer, and concentration is
40mM, pH 6.5.
Fourth aspect, the present invention provides a kind of detection sides FISH of histotomy for conventional FISH detection failure
Method, comprising the following steps:
1) microwave heating is carried out using histotomy of the pretreating reagent to routine FISH detection failure to add
Pressure processing；
2) using the histotomy of the detection failure of routine FISH described in pepsin digestion；
3) using the histocyte target gene site in FISH probe and the histotomy of routine FISH detection failure
Hybridization；
4) FISH probe of non-specific binding is eluted；
5) histotomy of the fluorescence microscope through above-mentioned steps treated routine FISH detection failure.
5th aspect, the present invention provides a kind of FISH detection reagents of histotomy for conventional FISH detection failure
Box, the kit include sodium citrate reagent and specification, and the specification indicates the work of sodium citrate salt buffer
Making concentration is 30-80mM, and work pH is 6.2-7.1.
Sodiocitrate
Sodiocitrate molecular formula C6H5Na3O7·2H2O。
Histotomy
" histotomy " refers to histotomy (histotomy, cell including narrow sense applied to FISH detection
Smear and chromosome sectioning), it is recognized that the fixed specimens paraffin embedding slices in 10% neutral formalin fixer, it is described
10% neutral formalin fix liquid making method are as follows: 10ml formalin+90ml 0.01mol/L PBS, with HCl adjust pH to
7.2-7.4。
Described " conventional FISH detection " is known, substantially attached drawing 1 to specifications for art technology
Method detected, and first step pretreating reagent be low concentration (particularly, refer to no more than 10mM；More particularly, refer to
10mM) and pH be no more than 6.0 citrate buffer." the detection failure " refers to when eliminating protease digestion
Between, under the premise of slice elution time and probe mass factor, FISH marking signal is weak or nothing or cell within a cell interstitial back
Scape causes by force very much to have covered FISH signal itself, so that the case where can not judging to result, wherein when protease digestion
Between, slice elution time and the method for removing of probe mass factor belong to routine techniques hand to those skilled in the art
Section, FISH marking signal is weak or the judgement of nothing or these too strong phenomenons of cell within a cell interstitial background is to those skilled in the art
It is also that can objectively evaluate for member." histotomy of conventional FISH detection failure " is mainly selected from following scenario described
It is any:
1) set time is more than 72 hours tissue blocks in 10% neutral formalin fixer；
2) tissue block of storage even more prolonged paraffin embedding more than 8 years；
3) cytoplasm and the very fine and close tumor tissue of cytoplasm, such as lung squamous cancer and certain sarcomas, but do not include
Lymthoma.
The pretreatment of histotomy in FISH detection
The pretreatment of histotomy includes following two step in FISH detection:
Step 1: citrate buffer heats histotomy.
Step 2: pepsin digestion histotomy.It normally, is the pepsin that 38.5U/ml is used at 37 DEG C or so
Handle histotomy 12-14min.
The invention has the advantages that:
It 1, should the present invention provides the pretreating reagent and method of a kind of histotomy for conventional FISH detection failure
Method improves the concentration and pH of sodium citrate salt buffer, this detect with conventional FISH used in concentration and pH difference compared with
Greatly, and it is different with the conventional means such as raising heat treatment temperature used by problems are solved, break to a certain extent
Habitual thinking mode, overcomes technology prejudice；
2, the present invention provides the sodium citrate salt buffers of four kinds of various concentrations and pH value, it was demonstrated that can detect that 71%-
73% with the routine FISH method of inspection failure example (tissue block or histotomy for being included in statistics have 302 cases, of all categories
Overlapping case in statistics has foreclosed), detection success rate significantly improves；
3, the pretreating reagent of the histotomy provided by the invention for routine FISH detection failure is sodiocitrate
Buffer is the standing reagent in laboratory, has advantage cheap, easy to use；
4, the invention demonstrates that concentration is 40mM, the sodium citrate salt buffer that pH is 6.5 is in 10% neutral formal
72 hours histotomies are fixed for more than in woods fixer to be particularly effective.
Detailed description of the invention
The simple process figure of Fig. 1 .FISH detection.
Fig. 2 needs Ewing ' the s sarcoma made a definite diagnosis, and 10% neutral formalin fixer is fixed for more than 72 hours；Using Chinese holly
After rafter acid sodium-salt buffer (80mM, pH7.1) pretreatment, nucleus is applied alone DAPI to dye, and display most cells core is still complete
It is whole.
Fig. 3 and Fig. 2 is that same Ewing ' the s sarcoma for needing to make a definite diagnosis is sliced, and 10% neutral formalin fixer is fixed
More than 72 hours；It after sodium citrate salt buffer (80mM, pH7.1) pretreatment, is marked with EWSR probe, shows EWSR base
Because 3 ' remote signalings (green) is separated with the fluorescent marker point of EWSR gene 5 ' end signal (red), can clarify a diagnosis as EWSR
Gene break transposition, and with multicopy phenomenon.
Fig. 4 adenocarcinoma of lung, 10% neutral formalin fixer are fixed for more than 72 hours, and conventional FISH examining report fails
Show signal；After sodium citrate salt buffer (40mM, pH6.5) pretreatment, ALK probe label display, although in cell
The background of visible green fluorescence in core, but still can clearly see letter in ALK3 ' remote signaling (red) and ALK gene
The fluorescent marker point of number (green) is overlapped, and can clarify a diagnosis does not have transversional mutant for ALK gene.
Fig. 5 adenocarcinoma of lung, 10% neutral formalin fixer fix 96 hours, and conventional FISH examining report fails to show
Signal；After sodium citrate salt buffer (60mM, pH6.8) pretreatment, ALK probe label display, although in cytoplasm
The background of interior visible green fluorescence, but still can clearly see signal in ALK3 ' remote signaling (red) and ALK gene
The fluorescent marker point of (green) separates, and can clarify a diagnosis and fracture and transversional mutant occurs for ALK gene.
Fig. 6 adenocarcinoma of lung, the paraffin pathological tissue block of storage 17 years, conventional FISH examining report fail to show signal；Using
After sodium citrate salt buffer (30mM, pH6.2) pretreatment, ALK probe label display, although visible green is glimmering in nucleus
The background of light, but still can clearly see the fluorescence of signal (green) in ALK3 ' remote signaling (red) and ALK gene
Mark point is overlapped, and can clarify a diagnosis does not have transversional mutant for ALK gene.
Fig. 7 adenocarcinoma of lung, the paraffin pathological tissue block of storage 28 years, conventional FISH examining report fail to show signal；Using
After sodium citrate salt buffer (80mM, pH7.1) pretreatment, ALK probe label display, although having in cytoplasm nucleus interstitial
Fluorescence background, but the label probe signal that still can clearly see in most cells core reaches analyzable quality,
The fluorescent marker point of signal (green) is overlapped in ALK3 ' remote signaling (red) and ALK gene in nucleus, and multicopy is presented
Phenomenon, can clarify a diagnosis does not have transversional mutant for ALK gene.
Fig. 8 acatalepsia, suspection are sarcomas, and the paraffin pathological tissue block of storage 32 years, conventional FISH examining report fails
Show signal；After sodium citrate salt buffer (80mM, pH7.1) pretreatment, EWSR probe label is selected, the results show that
Although having apparent artificial treatment pseudomorphism in nuclear targeting, 3 ' remote signaling of EWSR gene still can be clearly seen
The fluorescent marker point of (green) and EWSR gene 5 ' signal (red) is overlapped, and can clarify a diagnosis does not have transposition prominent for EWSR gene
Become.
Specific embodiment
Present inventor chances on the citric acid of higher concentration in an experiment reagent unexpectedly configuration error
Sodium salt buffer heat treatment slice, will can think originally the overlapping of the very fine and close reluctant certain sarcoma tissues of stubbornness
It is opened with crosslinking, and remaining tissue FISH signal is unexpectedly very clear, completes the present invention based on this.
It elaborates with reference to the accompanying drawing to specific embodiment provided by the invention.
The pretreating reagent sodium citrate salt buffer (one) of the invention of embodiment 1
Weigh sodiocitrate (CITRIC ACID TRISODIUM, Fisher Scientific, Cat#
AC327160010) 103.2 grams, it is configured to 1000ml 400mM stock solution, with 1N hydrochloric acid adjustment pH 7.0 or so, is added with preceding
ddH2It is 30mM that O, which adjusts concentration, is 6.2 with 1N hydrochloric acid adjustment pH.
The pretreating reagent sodium citrate salt buffer (two) of the invention of embodiment 2
Weigh sodiocitrate (CITRIC ACID TRISODIUM, Fisher Scientific, Cat#
AC327160010) 103.2 grams, it is configured to 1000ml 400mM stock solution, with 1N hydrochloric acid adjustment pH 7.0 or so, is added with preceding
ddH2It is 40mM that O, which adjusts concentration, is 6.5 with 1N hydrochloric acid adjustment pH.
The pretreating reagent sodium citrate salt buffer (three) of the invention of embodiment 3
Weigh sodiocitrate (CITRIC ACID TRISODIUM, Fisher Scientific, Cat#
AC327160010) 103.2 grams, it is configured to 1000ml 400mM stock solution, with 1N hydrochloric acid adjustment pH 7.0 or so, is added with preceding
ddH2It is 60mM that O, which adjusts concentration, is 6.8 with 1N hydrochloric acid adjustment pH.
The pretreating reagent sodium citrate salt buffer (four) of the invention of embodiment 4
Weigh sodiocitrate (CITRIC ACID TRISODIUM, Fisher Scientific, Cat#
AC327160010) 103.2 grams, it is configured to 1000ml 400mM stock solution, with 1N hydrochloric acid adjustment pH 7.0 or so, is added with preceding
ddH2It is 80mM that O, which adjusts concentration, is 7.1 with 1N hydrochloric acid adjustment pH.
The compliance test result of the pretreating reagent sodium citrate salt buffer of the invention of embodiment 5
One, experimental material and equipment
1) dimethylbenzene or dimethylbenzene substitute (Thermo ScientificTM ShandonTM Xylene
Substitute, Fisher Scientific, 99-905-07)；
2) micro-wave oven (medical or household)；
3) the dedicated pressure cooker of micro-wave oven (Nordic Ware Microwave Tender Cooker 2.5Quart, Asia
The inferior website of horse)；
4) sodiocitrate (CITRIC ACID TRISODIUM, Fisher sodiocitrate buffer stock: are weighed
Scientific, Cat#AC327160010) 103.2 grams, it is configured to 1000ml400mM stock solution and (is existed with 1N hydrochloric acid adjustment pH
7.0 or so).With preceding adjustment concentration as needed in 20mM-80mM, pH then is adjusted to desirable value with 1N hydrochloric acid again)；
5) pepsin (Pepsin from porcine gastric mucosa lyophilized powder, producer
Mark activity is 3,200-4,500units/mg protein, Sigma-Aldrich, Cat#P6887-1G), 1g is bis- in 50ml
It steams after thoroughly being dissolved in water, 50ml glycerol (molecular biology purity level, FISHERSCI, Cat#BP2291) magnetic agitation is added
After uniformly, Stock concentrations are that (be converted into active unit is 32-45units/ μ l to 10mg/ml, and mean value is 38.5units/ μ l.
Remarks: the active concentration examination criteria of active variant and each producer of the Unit Weight of each lot number of pepsin is not
One, the pre-detection test that digestion time is done to each lot number by each clinical labororatory is that the technology of FISH detection technique itself is wanted
Ask), it dispenses and to be stored in -20 DEG C of refrigerators stand-by；
6) pepsin digestion buffer (0.02N analyzes pure hydrochloric acid)；
7) 20 × SSC (from Fisher, stock solution, FISHERSCI, Cat#BP1325-4)
8) 2 × SSC buffer: using ten times of 20 × SSC of dilution of distilled water, adjusts pH in 7.2-7.5；
9) NP-40 (FISHSCI, Cat#NC9983875)；
10) 10ml NP-40 5%NP-40: is added to 200ml ddH2In O, magnetic stirrer over night；
11) in hybridization elution liquid I (0.5 × SSC/0.1%NP-40): 250ml 2 × SSC buffer, 20ml 5% is added
DdH is added in NP-40 solution2O to 1000ml, magnetic agitation mix；
12) in hybridization elution liquid II (2 × SSC/0.1%NP-40): 100ml 20 × SSC buffer, 20ml 5% is added
DdH is added in NP-40 solution2O to 1000ml, magnetic agitation mix；
13) probe (Abbott Molecular) proves (specific lot number is unlimited) through clinical detection:
Vysis EGFR/CEP 7FISH Probe Kit, Cat#01N35-020,
Vysis EWSR1Break Apart FISH Probe Kit, Cat#03N59-020,
Vysis FUS Break Apart FISH Probe Kit, Cat#03N58-020,
Vysis LSI ALK Break Apart Rearrangement Probe Kit, Cat#06N38-020；
14) all reagent water: pass through conventional high-pressure sterilization；
15) daily rubber mediate agent (Rubber Cement, Amazon)；
16) in situ hybridization instrument (Thermobrite StatSpin, Abbott Molecular)；
17) DAPI serves as a contrast dye liquor (VECTOR SHIELD MOUNTING MEDIA, Fisher Scientific, Cat#
NC9524612)；
18) microscopie unit: coming from CarlZess, and U.S. Metasystem assembles hardware and analysis software.
Two, experimental method
The concrete operation step of FISH detection are as follows:
1, slide pre-processes
1) using the slide of positive charge processing, paraffin section is prepared by routine clinical method, slice thickness is 4-5 microns；
2) slide is put into together in 90 DEG C of ovens together with glass frame, is baked piece 1 hour；
3) slide is taken out, puts it into the first cylinder dimethylbenzene substitute and dewaxes at room temperature 10 minutes；
4) it takes out slide and drains on filter paper to dripless and drip, put it into room temperature in the second cylinder dimethylbenzene substitute
Lower dewaxing 10 minutes；
5) it takes out slide and drains on filter paper to dripless and drip, put it into room temperature elution in the first cylinder dehydrated alcohol
Dimethylbenzene substitute 5 minutes；
6) it takes out slide and drains on filter paper to dripless and drip, put it into the second cylinder dehydrated alcohol, room temperature is washed
It is 10 minutes de-, the dimethylbenzene substitute of remaining is completely removed, is spontaneously dried in air, for use.
Above step 3) -6) it is carried out in chemical hood.
2, pretreatment before histotomy FISH label
7) it will be sliced for use and be placed in glass frame after the above dewaxing treatment, be then placed within the dedicated pressure cooker of micro-wave oven
Interior, any 1 liter of the pretreating reagent sodium citrate salt buffer of embodiment 1-4 is added, and (or liquid volume added to flood on slice completely
Subject to edge), it is necessary on inspection cover after pressure valve, in micro-wave oven use most high-grade heating, when can hear heating air valve it is faint
Stop heating (needing to grope heating time in advance) after outlet sound immediately, maintains pot cover to close 30 minutes under non-heated state
Afterwards, pot cover is opened, at room temperature natural cooling；
8) slice is taken out, at room temperature middle naturally dry；
9) slide is placed on back-to-back in glass frame, 37 DEG C of ovens preheat 5 minutes；
10) it takes above-mentioned 80 μ l pepsin to be added in the 80ml pepsin buffer of 37 DEG C of water-baths preheating, stirs evenly
And measuring temperature guarantees that actual temperature is at 37 DEG C in digestion fluid cylinder with electronics thermometer measure；
11) slice is immersed in pepsin digestion solution, clock reaction 12-14min (should pass through according to tissue types
Preliminary experiment determines the accurate reaction time)；
12) slice is immersed in 1 × PBS solution at room temperature and turns cylinder rapidly and washed 3 times；
13) slice is transferred to 70% ethanol solution and is dehydrated 1min at room temperature；
14) slice is transferred to 85% ethanol solution room temperature dehydration 1min；
15) slice is transferred to dehydrated alcohol room temperature dehydration 1min；
16) slice is taken out, is spontaneously dried at room temperature.
3, histotomy FISH hybridization and elution
17) writing of indictments, appeals, etc. is drawn with glass to iris out on slice to hybridization location；
18) add 5-10 μ l (according to the size of sample area) probe to hybridising region (bubble is avoided to generate)；
19) add clean coverslip, mediate agent with rubber and be sealed；
20) slice is put into situ hybridization instrument thermal station, is set to 94 DEG C of hybridization 5min；
21) slice is transferred to rapidly wet box, 42 DEG C of hybridized overnights (14 hours)；
22) it removes rubber and mediates agent, slice is immersed in 2 × SSC solution of 37 DEG C of water-baths preheating, it is light up and down every now and then
Piece is dripped in soft shaking, and coverslip is washed off；
22) slice is transferred in 0.4 × SSC/0.3%NP-40 solution of 75 DEG C of water-baths preheating and handles 3min, per minute
Piece is dripped in the soft shaking of front-rear direction；
23) it is transferred to 2 × SSC/0.1%NP-40 soaking at room temperature 3min；
24) slice, dark place natural air drying are taken out；
25) (15-20 μ l or so) DAPI lining dye liquor is dripped, hybridising region is arrived, coverslip is covered, to microscopy；
26) 63 under the dedicated microscope of FISH × oil under the microscope and photograph to record.
FISH detection is carried out to histotomy according to above step, the tissue block of test is in the received warp of clinical labororatory
The case of special requirement FISH detection consulting is sent after conventional FISH detection failure, therefore is selected without special organization type,
But lymphoma tissue is excluded except case statistics.
Three, experimental result
1, it is crossed for a long time in neutral formalin fixer with four kinds of pretreating reagent sodium citrate salt buffer detections
The statistical result of fixed tissue block
The statistics of this kind of case, it is desirable that while meeting the following conditions:
1) record shows, group is woven in 10% neutral formalin fixer and is fixed for more than 72 hours；
2) routine FISH detects preconditioning recipe (sodium citrate salt buffer, 10mM, pH6.0) detection failure, and arranges
Removing protein enzymic digestion time, the factor for being sliced elution time and probe mass；
3) resting period of paraffin pathology block and dense structure's degree are unlimited.
It the results are shown in Table 1.
Table 1
Conclusion: any one sodiocitrate buffer formulation of Application Example 1-4 has certain detection success rate, makes
Originally preprocess method cannot show that the case of FISH signal is resolved, and total success rate is 73.3%.Wherein 40mM, pH6.5
Sodium citrate salt buffer assembly power be up to 84.6%.
Fig. 2-Fig. 5 is FISH detection example.
2, the analysis for the paraffin pathological tissue block stored for a long time with four kinds of pretreating reagent sodium citrate salt buffer detections
As a result
The statistics of this kind of case, it is desirable that while meeting the following conditions:
1) record shows that the pathology paraffin mass resting period is more than 8 years or more；
2) routine FISH detects preconditioning recipe (sodium citrate salt buffer, 10mM, pH6.0) detection failure, and arranges
Removing protein enzymic digestion time, the factor for being sliced elution time and probe mass；
3) formalin set time and dense structure's degree are unlimited；
4) select four kinds of sodiocitrate buffer formulations of embodiment 1-4 unlimited.
It the results are shown in Table 2.
Table 2
Conclusion: the summation of four last testing results of formula of Application Example 1-4 shows to each resting period group
Tissue block can be such that certain case detects successfully；And age shorter tissue is stored, the ratio being resolved is higher.
Fig. 6-Fig. 7 is FISH detection example.
3, the tumour very fine and close with four kinds of pretreating reagent sodium citrate salt buffer detection cytoplasm and cytoplasm
The result of tissue block
The statistics of this kind of case, it is desirable that while meeting the following conditions:
1) routine FISH detects preconditioning recipe (sodium citrate salt buffer, 10mM, pH6.0) and failure is checked or analyzed,
And the factor for excluding the protease digestion time, being sliced elution time and probe mass；
2) the reason of failure be as cytoplasm and cytoplasm it is very fine and close caused by between protein and protein or
Crosslinking between person's protein and nucleic acid is excessively fine and close, so that probe is unable to reach the site of target gene or can not be integrated to target gene
Site, or due between cell and intracellular background fluorescence has covered the specific signals of FISH；
3) formalin set time and pathology paraffin mass resting period are unlimited.
It the results are shown in Table 3.
Table 3
Conclusion: certain case can be made to obtain detecting successfully using four formulas of 1-4；But concentration is higher, although
The form of nucleus can completely show, but the prototype structure between histocyte change to think change it is also bigger.
Fig. 8 is FISH detection example.
4, with the result of four kinds of pretreating reagent sodium citrate salt buffer detection lymphoma tissue slices
The FISH detection of lymphoma tissue slice slice totally 28, be conventional FISH detection (sodium citrate salt buffer,
10mM, pH6.0) failure after (mainly signal it is too weak or nuclear area be overlapped, the signal of each nucleus can not be read
Copy number), the case of special FISH detection consulting is sent, after four kinds of preprocess methods of Application Example 1-4, as a result not only
Do not improve, eucaryotic cell structure is not also improved by different degrees of damage, signal quality instead, or even loses.
Therefore, preprocess method of the invention is completely unsuitable for the histotomy of lymthoma, therefore does not also enumerate
Among statistical table.
Four, conclusion
The present invention provides four kinds of new sodium citrate salt buffer preconditioning recipes, can solve routine clinical FISH inspection
The case for the 71%-73% that dendrometry loses.It is important to note that although the sodiocitrate buffer formulation (30- of high concentration
80mM) there is certain artificial shadow to histiocytic structure than the sodiocitrate buffer formulation (10mM) of general clinical application
The factor of sound, but the emphasis of FISH detection is the change of target gene on the endonuclear chromosome of observation, as long as DNA serves as a contrast stain
DAPI shows that the integrality of nucleus, the FISH signal detected is clear, internal reference signal is correct.In combination cell division
Phase chromosome FISH testing result, in the case tested, 71%-73% is the result is that believable.
It should be noted that inventor suggests locating FISH histotomy in advance using pretreating reagent of the invention
Reason, preferably holds following principle and points for attention:
1) after examining failure for conventional sodiocitrate buffer formulation (10mM, pH6.0), it need to confirm and eliminate albumen
After the improper factor and probe mass factor of enzymic digestion and elution process, pretreating reagent citric acid of the invention just can be used
Sodium salt buffer formulation；
2) in use, suggesting beginning trying from the low-concentration formulations of embodiment 1, the signal of approval is detected；If
The desired result not obtained can attempt the formula of any higher concentration of embodiment 2-4.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (9)
1. a kind of pretreating reagent of the histotomy for conventional FISH detection failure, which is characterized in that the pretreatment
Reagent is sodium citrate salt buffer, and the concentration and pH of the sodium citrate salt buffer are respectively selected from appointing in following each group
One group:
A) 30mM, pH6.2；
B) 40mM, pH6.5；
C) 60mM, pH6.8；
D) 80mM, pH7.1.
2. pretreating reagent according to claim 1, which is characterized in that the tissue of the conventional FISH detection failure is cut
Piece is the fixed paraffin-embedded tissue slice of 10% neutral formalin fixer, and be eliminate the protease digestion time,
Failure is detected under the premise of being sliced elution time and probe mass factor.
3. application of the pretreating reagent of any of claims 1 or 2 in FISH detection.
4. application according to claim 3, which is characterized in that the application is using pre- described in as claimed in claim 1 or 22
Reagent treatment carries out microwave heating pressurized treatments to the histotomy of conventional FISH detection failure.
5. a kind of preprocess method of the histotomy for conventional FISH detection failure, which is characterized in that the method packet
The histotomy to routine FISH detection failure containing pretreating reagent described in using as claimed in claim 1 or 22 carries out microwave and adds
The step of hot pressurized treatments.
6. preprocess method according to claim 5, which is characterized in that the preprocess method specifically: will be described
The histotomy of conventional FISH detection failure is placed in the dedicated pressure cooker of micro-wave oven, and the pretreating reagent is added and makes
Flood histotomy upper limb completely, heat in micro-wave oven, stop immediately after can hear heating air valve faint outlet sound
It only heats, after maintaining high-pressure cooker cover to close 25-30 minutes under non-heated state, opens pot cover, at room temperature natural cooling.
7. preprocess method according to claim 5, which is characterized in that the histotomy of the routine FISH detection failure
To be fixed for more than 72 hours paraffin-embedded tissue slices in 10% neutral formalin fixer, and excluding protease digestion
Failure is detected under the premise of time, slice elution time and probe mass factor；The pretreating reagent is sodiocitrate
Buffer, concentration 40mM, pH 6.5.
8. a kind of FISH detection method of the histotomy for conventional FISH detection failure, which is characterized in that including following step
It is rapid:
1) it is carried out using histotomy of the pretreating reagent described in as claimed in claim 1 or 22 to routine FISH detection failure micro-
Wave heating pressurized treatments；
2) using the histotomy of the detection failure of routine FISH described in pepsin digestion；
3) hybridized using the histocyte target gene site in FISH probe and the histotomy of routine FISH detection failure；
4) FISH probe of non-specific binding is eluted；
5) histotomy of the fluorescence microscope through above-mentioned steps treated routine FISH detection failure.
9. a kind of FISH detection kit of the histotomy for conventional FISH detection failure, which is characterized in that the examination
Agent box includes sodium citrate reagent and specification, and the specification indicates working concentration and the work of sodium citrate salt buffer
Any group in following each group of pH:
A) 30mM, pH6.2；
B) 40mM, pH6.5；
C) 60mM, pH6.8；
D) 80mM, pH7.1.
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