CN105861659A - Pretreatment liquid and pretreatment method used for fluorescence in situ hybridization of FFPE tissue slices - Google Patents
Pretreatment liquid and pretreatment method used for fluorescence in situ hybridization of FFPE tissue slices Download PDFInfo
- Publication number
- CN105861659A CN105861659A CN201610226528.XA CN201610226528A CN105861659A CN 105861659 A CN105861659 A CN 105861659A CN 201610226528 A CN201610226528 A CN 201610226528A CN 105861659 A CN105861659 A CN 105861659A
- Authority
- CN
- China
- Prior art keywords
- pretreatment fluid
- tissue slice
- fluorescence
- situ hybridization
- pretreatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the biotechnical field, and concretely relates to a pretreatment method and a pretreatment liquid used for fluorescence in situ hybridization of FFPE tissue slices. The pretreatment liquid comprises a pretreatment liquid I and a pretreatment liquid II; the component of the pretreatment I is alkylphenol polyoxyethylene (TX-10) and/or octylphenoxypolyethoxyethanol; and components of the pretreatment liquid II comprise alkylphenol polyoxyethylene (TX-10) and/or octylphenoxypolyethoxyethanol, and also comprise ethylene diamine tetraacetic acid (EDTA) and/or a citric acid buffer. Paraffin is directly dissolved and emulsified in aqueous environment with the temperature being higher than the melting point of the paraffin through the pretreatment liquid I and the pretreatment liquid II by using the hydrophilic and lipophilic characteristics of an emulsifier in order to complete hydration and permeation, so steps are simplified, and time is saved. The pretreatment liquid II also can destroy formalin fixation induced cross-linking among proteins, so the FFPE tissues can be easily digested by proteases, and the autofluorescence and the background of the tissues are substantially reduced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of pretreatment side for fluorescence in situ hybridization FFPE tissue slice
Method and pretreatment fluid.
Background technology
Fluorescence in situ hybridization is that a kind of one grown up in radioactive in situ hybridization technical foundation in the eighties in 20th century is non-puts
Penetrating property molecular genetic techniques, a kind of new in-situ hybridization method formed so that fluorescein labelling replaces labelled with radioisotope.
FISH has safe, quick, highly sensitive and shows the advantages such as multiple color simultaneously.
Carry out fluorescence in situ hybridization experiment time use traditional method typically require FFPE sample slice is dewaxed, rehydration,
Hydrogen thiocyanate receives process, protease digestion, 2 × SSC rinsing and dehydration of alcohol drying and other steps, and omnidistance step is complicated and the longest
(Fig. 1), needing to use the toxic reagent such as dimethylbenzene and sodium rhodanate in the most traditional processing method, this has all been significantly greatly increased reality
Environment is caused severe contamination by health risk and the experiment garbage of testing operator.
Additionally, the autofluorescence that tissue itself produces in fluorescence in situ hybridization is tested also produces the biggest doing to experimental result
Disturb, when especially probe fragment labelling is less;These autofluorescences are generally by the endogenous riboflavin of histiocyte, lipofuscin, net
Shape fiber, collagen protein and elastin laminin etc. cause.The histiocyte vigorous to some metabolism or exist a large amount of collagen protein and
When the tissue of elastin laminin or reticular fiber carries out fluorescence in situ hybridization, its results of hybridization presents stronger oneself under fluorescence microscope
Fluoresce and background, have a strong impact on result interpretation, and traditional processing method not can solve this problem.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is therefore intended that provide a kind of pre-for fluorescence in situ hybridization FFPE tissue slice
Processing method and pretreatment fluid.
For achieving the above object, the technical solution used in the present invention is:
A kind of pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice, including pretreatment fluid I and pretreatment fluid II.
In such scheme, described pretreatment fluid I is emulsifier solution, and described emulsifying agent is alkylphenol polyoxyethylene (TX-10)
And/or Nonidet P40.
In such scheme, the component of described pretreatment fluid II includes that emulsifying agent, described emulsifying agent are alkylphenol polyoxyethylene
And/or Nonidet P40 (TX-10).
In such scheme, in described pretreatment fluid I and pretreatment fluid II, the mass concentration of emulsifying agent is 0.1~10%.
In such scheme, the component of described pretreatment fluid II also includes that other components, other components described are ethylenediaminetetraacetic acid
And/or citrate buffer solution (EDTA).
In such scheme, the described ethylenediaminetetraacetic acid (EDTA) concentration in pretreatment fluid II is 10~100mM, described
Citrate buffer solution concentration in pretreatment fluid II is 5~100mM, pH value 4.8~6.2.
A kind of preprocess method for fluorescence in situ hybridization FFPE tissue slice, comprises the steps:
(1) roasting sheet: tissue slice is placed on the roasting sheet machine of 65 DEG C roasting sheet 3~5min;
(2) pretreatment fluid I process: tissue slice is immersed and is preheated to 5min in the pretreating agent I of 68 DEG C;
(3) pretreatment fluid II process: take out the tissue slice in pretreatment fluid I, drains the pretreatment of remnants in absorbent paper
Liquid I, then tissue slice immersion is preheated to 10min in the pretreatment fluid II of 72 DEG C, shake slide up and down once every 2~3min;
(4) tissue slice is immersed in be preheated in the deionized water of 68 DEG C wash 30s;
(5) take out tissue slice and immerse in the pepsin working solution being preheated to 37 DEG C now joined, digesting 5~15min;
(6) after completing digestion, tissue slice is immersed 1min in 2 × SSC solution;
(7) repeat step (6), then take out tissue slice and pass sequentially through 70Vt% (volumetric concentration), 85Vt%, 100Vt%
Ethanol each 2min serial dehydration;
(8) naturally dry after taking out tissue slice, test for follow-up fluorescence in situ hybridization.
In such scheme, the component of described pepsin working solution includes: 2wt% pepsin, 0.02M HCL, described stomach egg
The enzyme of white enzyme is lived as 3500U/mg.
In such scheme, the pH of described 2 × SSC solution is 6.8~7.2.
In preparation method of the present invention, in step (5), digestible degree can be passed through at common light field basis of microscopic observation,
If digestion deficiency, will need section to be immersed again Digestive system digests several minutes, again observe;If digestion is excessively, should again enter
Row experiment.
In preparation method of the present invention, step (2) and (3) jointly complete paraffin section de-waxing, rehydration and break albumen
Cross-link the effect of penetrating tissue.
Beneficial effects of the present invention is as follows: (1) present invention employs pretreatment fluid I and pretreatment fluid II higher than paraffin melting point
The feature utilizing the hydrophilic oleophylic again of emulsifying agent under aqueous environments is directly dissolved emulsified wax and completes aquation and penetrating work
With, simplify traditional hydrodewaxing step and save the time (the process time was shortened to 40~50 minutes by traditional 2.5 hours).
(2) present invention use pretreatment fluid II can also break formaldehyde fix between the albumen caused cross-link so that FFPE organize more
Easily by protease digestion, can significantly reduce autofluorescence and the background of tissue.(3) present invention has broken away from traditional dewaxing agent
Dimethylbenzene, whole operating process is nontoxic and free from extraneous odour is without carrying out in fume hood;The present invention simplify operating procedure and
The waste liquid that the dewaxing agent of aqueous is more suitable for self-reacting device operation and experiment generation is environmentally friendly pollution-free.
Accompanying drawing explanation
Fig. 1 is Several Traditional Preconditioning Methods and preprocess method provided by the present invention time-consumingly contrasts.
Fig. 2 is for using pretreatment fluid of the present invention and preprocess method to process breast carcinoma FFPE tissue samples, and uses Vysis
HER2 gene amplification detection kit carries out hybridization in situ experiment, section knot of mounting microscopy after washing stringency for detection probe
Really.
Fig. 3 is for using Several Traditional Preconditioning Methods to process breast carcinoma FFPE tissue samples, and uses Vysis HER2 gene amplification
Detection kit carries out hybridization in situ experiment, section result of mounting microscopy after washing stringency for detection probe.
Fig. 4 is for using pretreatment fluid of the present invention and preprocess method to process pulmonary carcinoma FFPE tissue samples, and uses Vysis
ALK fusion gene detection kit carries out hybridization in situ experiment, section knot of mounting microscopy after washing stringency for detection probe
Really.
Fig. 5 is for using Several Traditional Preconditioning Methods to process breast carcinoma FFPE tissue samples, and uses ALK fusion gene detection examination
Agent box carries out hybridization in situ experiment, section result of mounting microscopy after washing stringency for detection probe.
Detailed description of the invention
In order to be more fully understood that the present invention, it is further elucidated with present disclosure below in conjunction with embodiment, but present disclosure is not
It is limited only to the following examples.
Embodiment 1
The present embodiment chooses breast carcinoma FFPE tissue samples as test sample, uses preprocess method of the present invention to carry out pre-
Process, comprise the steps:
(1) roasting sheet: tissue slice is placed on the roasting sheet machine of 65 DEG C roasting sheet 3~5min;
(2) pretreatment fluid I process: tissue slice is immersed and is preheated to 5min in the pretreating agent I of 68 DEG C;Described pretreatment
Liquid I composition is 1wt% Nonidet P40 and 0.5wt% alkylphenol polyoxyethylene.
(3) pretreatment fluid II process: take out the tissue slice in pretreatment fluid I, drains the pretreatment of remnants in absorbent paper
Liquid I, then tissue slice immersion is preheated to 10min in the pretreatment fluid II of 72 DEG C, shake slide up and down once every 2~3min;
Described pretreatment fluid II composition is 0.5wt% Nonidet P40 and 10mM citrate buffer solution (pH 6.2).
(4) tissue slice is immersed in be preheated in the deionized water of 68 DEG C wash 30s;
(5) take out tissue slice and immerse pepsin working solution (2% pepsin being preheated to 37 DEG C now joined
(3500U/mg), 0.02M HCL) in, digest 5~15min;
(6) after completing digestion, tissue slice is immersed 1min in 2 × SSC solution (PH=6.8~7.2);
(7) repeat step (6), take out tissue slice and pass sequentially through 70%, 85%, 100% ethanol each 2min serial dehydration;
(8) naturally dry after taking out tissue slice, test for follow-up fluorescence in situ hybridization.
Use Vysis HER2 gene amplification detection kit for detection probe, dry mammary gland pretreated to the present embodiment
Cancer FFPE tissue slice carries out fluorescence in situ hybridization, and concrete operation step is as follows:
(1) take the section of pretreated dry FFPE tissue samples, add 10uL above-mentioned Vysis HER2 gene amplification inspection
Test agent box is detection probe, covered with rubber glue mounting;
(2) section after mounting is placed in hybridization instrument hybridization procedures is set: 83 DEG C of degeneration 5min, 37 DEG C of hybridized overnight;
(3) take out the slide of Overnight hybridization, carefully tear rubber glue off and take off lid fragmentation gently, slide is immersed and is preheated to 65 DEG C
Cleaning mixture in (0.3%NP40,0.4 × SSC) washing 5min;
(4) take out slide, immerse washing 2~3min in the rinsing liquid (0.1%NP40,2 × SSC) of room temperature;
(5) immersing after taking out slide and wash 2~3min in 70% ethanol, after taking-up, room temperature is dried;Fluorescence microscopy Microscopic observation breast
The experimental result of adenocarcinoma FFPE tissue samples, analyte signal intensity and background are as shown in Figure 2.
Comparative example 1
Choose breast carcinoma FFPE tissue samples as test sample, use and be traditionally used for fluorescence in situ hybridization FFPE tissue and cut
The preprocess method of sheet carries out pretreatment, comprises the steps:
(1) roasting sheet: tissue is placed at 65 DEG C and overnight toasts;
(2) dewaxing: tissue slice is immersed room temperature dewaxing 2 times in dimethylbenzene, each 15 minutes;
(3) by section immersion 100% ethanol 5 minutes;
(4) 100% ethanol will be placed under tissue slice successively room temperature, 85% ethanol, each 2 minutes rehydrations in 70% ethanol;
(5) tissue slice room temperature is immersed deionized water 3 minutes, dry with the examination of lint-free napkin;
(6) tissue slice is placed in 20min in 80 DEG C of 0.1M HCL solution;
Tissue slice is processed 30 minutes with 5% (w/v) sodium rhodanate at (7) 50 DEG C;
(8) in 2XSSC solution, 2 times are rinsed under room temperature, each 5 minutes;
(9) take 0.4ml E.C. 3.4.21.64 to store liquid (20mg/ml) and be dissolved in 40ml 2XSSC and obtain E.C. 3.4.21.64 working solution
(200 μ g/ml), immerses tissue slice in E.C. 3.4.21.64 working solution 37 DEG C and hatches 20~30 minutes;
(10) tissue slice is after E.C. 3.4.21.64 processes, rinsing 2 times in 2XSSC, each 5 minutes;
(11) tissue slice is immersed in 0.1M HCL soaking at room temperature 5~10 minutes;
(12) in 2XSS solution, 2 times are rinsed, each 5 minutes;
(13) tissue section slide is immersed successively 70%, 85%, dehydration in each 2 minutes in 100% ethanol;
(14) natural drying slide.
Using Vysis HER2 gene amplification detection kit is detection probe, to using pretreatment traditional described in comparative example 1
The pretreated dry breast carcinoma FFPE tissue slice of method carries out fluorescence in situ hybridization, and concrete operation step is as follows:
(1) take pretreated dry sliced, add 10ul above-mentioned Vysis HER2 gene amplification detection kit for inspection
Probing pin, covered with rubber glue mounting;
(2) section after mounting is placed in hybridization instrument hybridization procedures is set: 83 DEG C of degeneration 5min, 37 DEG C of hybridized overnight;
(3) take out the slide of Overnight hybridization, carefully tear rubber glue off and take off lid fragmentation gently, slide is immersed and is preheated to 65 DEG C
Cleaning mixture in (0.3%NP40,0.4 × SSC) washing 5min;
(4) take out slide, immerse washing 2~3min in the rinsing liquid (0.1%NP40,2 × SSC) of room temperature;
(5) immersing after taking out slide and wash 2~3min in 70% ethanol, after taking-up, room temperature is dried;Fluorescence microscopy Microscopic observation breast
The experimental result of adenocarcinoma FFPE tissue samples, analyte signal intensity and background are as shown in Figure 3.
Embodiment 1 will use the experiment obtained for fluorescence in situ hybridization FFPE tissue slice preprocess method of the present invention
The experimental result (Fig. 3) using traditional preprocess method to obtain in result (Fig. 2) and comparative example 1 compares, permissible
Find out: use breast carcinoma FFPE after fluorescence in situ hybridization FFPE tissue slice preprocess method processes of the present invention
Tissue samples, is carried out fluorescence in situ hybridization, and autofluorescence and the background of its tissue are lower, and signaling point is clear, signal interpretation
It is easier to.
Embodiment 2
The present embodiment chooses adenocarcinoma of lung FFPE tissue samples as test sample, uses of the present invention for fluorescence in situ hybridization
FFPE tissue slice preprocess method carries out pretreatment, comprises the steps:
(1) roasting sheet: tissue slice is placed on the roasting sheet machine of 65 DEG C roasting sheet 3~5min;
(2) pretreatment fluid I process: tissue slice is immersed and is preheated to 5min in the pretreating agent I of 68 DEG C;Described pretreatment
Liquid I is 1wt% Nonidet P40 and 0.5wt% alkylphenol polyoxyethylene.
(3) pretreatment fluid II process: take out the tissue slice in pretreatment fluid I, drains the pretreatment of remnants in absorbent paper
Liquid I, then tissue slice immersion is preheated to 10min in the pretreatment fluid II of 72 DEG C, shake slide up and down once every 2~3min;
Described pretreatment fluid II composition is 0.5wt% Nonidet P40,10mM citrate buffer solution (pH 6.0) and 10mM
EDTA。
(4) tissue slice is immersed in be preheated in the deionized water of 68 DEG C wash 30s;
(5) take out tissue slice and immerse pepsin working solution (2% pepsin being preheated to 37 DEG C now joined
(3500U/mg), 0.02M HCL) in, digest 5~15min;
(6) after completing digestion, tissue slice is immersed 1min in 2 × SSC solution (PH=6.8~7.2);
(7) repeat step (6), take out tissue slice and pass sequentially through 70%, 85%, 100% ethanol each 2min serial dehydration;
(8) naturally dry after taking out tissue slice, test for follow-up fluorescence in situ hybridization.
Use Vysis ALK fusion gene detection kit for detection probe, dry adenocarcinoma of lung pretreated to the present embodiment
FFPE tissue slice carries out fluorescence in situ hybridization, and concrete operation step is as follows:
(1) take pretreated dry sliced, add 10uL above-mentioned Vysis ALK fusion gene detection kit for detection visit
Pin, covered with rubber glue mounting;
(2) section after mounting is placed in hybridization instrument hybridization procedures is set: 83 DEG C of degeneration 5min, 37 DEG C of hybridized overnight;
(3) take out the slide of Overnight hybridization, carefully tear rubber glue off and take off lid fragmentation gently, slide is immersed and is preheated to 65 DEG C
Cleaning mixture in (0.3%NP40,0.4 × SSC) washing 5min;
(4) take out slide, immerse washing 2~3min in the rinsing liquid (0.1%NP40,2 × SSC) of room temperature;
(5) immersing after taking out slide and wash 2~3min in 70% ethanol, after taking-up, room temperature is dried;Fluorescence microscopy Microscopic observation lung
The experimental result of adenocarcinoma FFPE tissue samples, analyte signal intensity and background are as shown in Figure 4.
Comparative example 2
Choose adenocarcinoma of lung FFPE tissue samples as test sample, use and be traditionally used for fluorescence in situ hybridization FFPE tissue and cut
The preprocess method of sheet carries out pretreatment, comprises the steps:
(1) roasting sheet: tissue is placed at 65 DEG C and overnight toasts;
(2) dewaxing: tissue slice is immersed room temperature dewaxing 2 times in dimethylbenzene, each 15 minutes;
(3) by section immersion 100% ethanol 5 minutes;
(4) 100% ethanol will be placed under tissue slice successively room temperature, 85% ethanol, each 2 minutes rehydrations in 70% ethanol;
(5) tissue slice room temperature is immersed deionized water 3 minutes, dry with the examination of lint-free napkin;
(6) tissue slice is placed in 20min in 80 DEG C of 0.1M HCL solution;
Tissue slice is processed 30 minutes with 5% (w/v) sodium rhodanate at (7) 50 DEG C;
(8) in 2XSSC solution, 2 times are rinsed under room temperature, each 5 minutes;
(9) take 0.4ml E.C. 3.4.21.64 to store liquid (20mg/ml) and be dissolved in 40ml 2XSSC and obtain E.C. 3.4.21.64 working solution
(200 μ g/ml), immerses tissue slice in E.C. 3.4.21.64 working solution 37 DEG C and hatches 20~30 minutes;
(10) tissue slice is after E.C. 3.4.21.64 processes, rinsing 2 times in 2XSSC, each 5 minutes;
(11) tissue slice is immersed in 0.1M HCL soaking at room temperature 5~10 minutes;
(12) in 2XSS solution, 2 times are rinsed, each 5 minutes;
(13) tissue section slide is immersed successively 70%, 85%, dehydration in each 2 minutes in 100% ethanol;
(14) natural drying slide.
Using Vysis ALK fusion gene detection kit is detection probe, to using pretreatment side traditional described in comparative example 2
The pretreated dry adenocarcinoma of lung FFPE tissue slice of method carries out fluorescence in situ hybridization, and concrete operation step is as follows:
(1) take pretreated dry sliced, add 10uL above-mentioned Vysis ALK fusion gene detection kit for detection visit
Pin, covered with rubber glue mounting;
(2) section after mounting is placed in hybridization instrument hybridization procedures is set: 83 DEG C of degeneration 5min, 37 DEG C of hybridized overnight;
(3) take out the slide of Overnight hybridization, carefully tear rubber glue off and take off lid fragmentation gently, slide is immersed and is preheated to 65 DEG C
Cleaning mixture in (0.3%NP40,0.4 × SSC) washing 5min;
(4) take out slide, immerse washing 2~3min in the rinsing liquid (0.1%NP40,2 × SSC) of room temperature;
(5) immersing after taking out slide and wash 2~3min in 70% ethanol, after taking-up, room temperature is dried;Fluorescence microscopy Microscopic observation lung
The experimental result of adenocarcinoma FFPE tissue samples, analyte signal intensity and background are as shown in Figure 5.
Embodiment 3 will use the experiment obtained for fluorescence in situ hybridization FFPE tissue slice preprocess method of the present invention
The experimental result (Fig. 5) using traditional preprocess method to obtain in result (Fig. 4) and comparative example 2 compares, permissible
Find out: use breast carcinoma FFPE after fluorescence in situ hybridization FFPE tissue slice preprocess method processes of the present invention
Tissue samples, autofluorescence and the background of its tissue are lower, and signaling point is clear, signal interpretation is easier to.
Obviously, above-described embodiment is only by clearly demonstrating made example, and not restriction to embodiment.For institute
For the those of ordinary skill in genus field, can also make other changes in different forms on the basis of the above description.
Here without also cannot all of embodiment be given exhaustive.And the obvious change therefore amplified or variation still in
Within the protection domain of the invention.
Claims (9)
1. the pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice, it is characterised in that include pretreatment fluid I and pretreatment fluid II.
Pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice the most according to claim 1, it is characterised in that described pretreatment fluid I is emulsifier solution, described emulsifying agent is alkylphenol polyoxyethylene and/or Nonidet P40.
Pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice the most according to claim 1, it is characterised in that the component of described pretreatment fluid II includes that emulsifying agent, described emulsifying agent are alkylphenol polyoxyethylene and/or Nonidet P40.
Pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice the most according to claim 1, it is characterised in that in pretreatment fluid I and pretreatment fluid II, the mass concentration of emulsifying agent is 0.1 ~ 10%.
Pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice the most according to claim 3, it is characterised in that the component of described pretreatment fluid II also includes that other components, other components described are ethylenediaminetetraacetic acid and/or citrate buffer solution.
Pretreatment fluid for fluorescence in situ hybridization FFPE tissue slice the most according to claim 5, it is characterized in that, described ethylenediaminetetraacetic acid concentration in pretreatment fluid II is 10 ~ 100mM, and described citrate buffer solution concentration in pretreatment fluid II is 5 ~ 100mM, pH value 4.8 ~ 6.2.
7. the arbitrary described pretreatment fluid of claim 1 ~ 6 is for the preprocess method of fluorescence in situ hybridization FFPE tissue slice, it is characterised in that comprise the steps:
(1) roasting sheet: tissue slice is placed on the roasting sheet machine of 65 DEG C roasting sheet 3 ~ 5min;
(2) pretreatment fluid I process: tissue slice is immersed and is preheated to 5min in the pretreating agent I of 68 DEG C;
(3) pretreatment fluid II process: take out the tissue slice in pretreatment fluid I, drains the pretreatment fluid I of remnants in absorbent paper, then tissue slice immersion is preheated to 10min in the pretreatment fluid II of 72 DEG C, shake slide up and down once every 2 ~ 3min;
(4) tissue slice is immersed in be preheated in the deionized water of 68 DEG C wash 30s;
(5) take out tissue slice and immerse in the pepsin working solution being preheated to 37 DEG C now joined, digesting 5 ~ 15min;
(6) after completing digestion, tissue slice is immersed 1min in 2 × SSC solution;
(7) repeating step (6), then taking out tissue slice and passing sequentially through volumetric concentration is 70%, 85%, 100% ethanol each 2min serial dehydration;
(8) naturally dry after taking out tissue slice, test for follow-up fluorescence in situ hybridization.
Preprocess method the most according to claim 7, it is characterised in that the component of described pepsin working solution includes: 2wt% pepsin, 0.02M HCL, described pepsic enzyme is lived as 3500U/mg.
Preprocess method the most according to claim 7, it is characterised in that the pH of described 2 × SSC solution is 6.8 ~ 7.2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610226528.XA CN105861659A (en) | 2016-04-13 | 2016-04-13 | Pretreatment liquid and pretreatment method used for fluorescence in situ hybridization of FFPE tissue slices |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610226528.XA CN105861659A (en) | 2016-04-13 | 2016-04-13 | Pretreatment liquid and pretreatment method used for fluorescence in situ hybridization of FFPE tissue slices |
Publications (1)
Publication Number | Publication Date |
---|---|
CN105861659A true CN105861659A (en) | 2016-08-17 |
Family
ID=56637746
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610226528.XA Pending CN105861659A (en) | 2016-04-13 | 2016-04-13 | Pretreatment liquid and pretreatment method used for fluorescence in situ hybridization of FFPE tissue slices |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105861659A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107560918A (en) * | 2017-09-22 | 2018-01-09 | 漯河医学高等专科学校 | A kind of pathological tissue specimen fixer |
CN109975095A (en) * | 2019-02-22 | 2019-07-05 | 嘉兴雅康博医学检验所有限公司 | A kind of pretreatment liquid and pre-treating method of fluorescence in situ hybridization |
WO2023206356A1 (en) * | 2022-04-29 | 2023-11-02 | 京东方科技集团股份有限公司 | Reagent and detection method for fluorescence in situ hybridization |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002023156A1 (en) * | 2000-09-15 | 2002-03-21 | Biogenex Laboratories | Enhancement of in situ hybridization |
CN103409505A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting BCR/ABL fusion gene free from repetitive sequence |
CN104334717A (en) * | 2012-01-23 | 2015-02-04 | 理查艾伦科学公司 | Dewaxing buffer containing a water-soluble organic solvent and methods of use thereof |
CN105164259A (en) * | 2013-02-25 | 2015-12-16 | 拜奥卡蒂斯股份有限公司 | Isolation of nucleic acids |
-
2016
- 2016-04-13 CN CN201610226528.XA patent/CN105861659A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002023156A1 (en) * | 2000-09-15 | 2002-03-21 | Biogenex Laboratories | Enhancement of in situ hybridization |
CN104334717A (en) * | 2012-01-23 | 2015-02-04 | 理查艾伦科学公司 | Dewaxing buffer containing a water-soluble organic solvent and methods of use thereof |
CN105164259A (en) * | 2013-02-25 | 2015-12-16 | 拜奥卡蒂斯股份有限公司 | Isolation of nucleic acids |
CN103409505A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting BCR/ABL fusion gene free from repetitive sequence |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107560918A (en) * | 2017-09-22 | 2018-01-09 | 漯河医学高等专科学校 | A kind of pathological tissue specimen fixer |
CN109975095A (en) * | 2019-02-22 | 2019-07-05 | 嘉兴雅康博医学检验所有限公司 | A kind of pretreatment liquid and pre-treating method of fluorescence in situ hybridization |
WO2023206356A1 (en) * | 2022-04-29 | 2023-11-02 | 京东方科技集团股份有限公司 | Reagent and detection method for fluorescence in situ hybridization |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2018250513B2 (en) | Two phase immiscible system for the pretreatment of embedded biological samples | |
CN105861659A (en) | Pretreatment liquid and pretreatment method used for fluorescence in situ hybridization of FFPE tissue slices | |
US20030175852A1 (en) | Ehancement of in situ hybridization | |
Renshaw | Immunohistochemistry and immunocytochemistry | |
DE69532224D1 (en) | DEPARAFFINIZING COMPOSITIONS AND METHOD FOR USE THEREOF | |
Uddin et al. | Physical and biochemical characterization of chemically treated pollen shells for potential use in oral delivery of therapeutics | |
CN103940658A (en) | Method for manufacturing paraffin-embedded tissue cell specimen | |
CN110257483B (en) | Hybridization solution for in situ hybridization, preparation method thereof and detection kit | |
CN107843470A (en) | A kind of preparation method of Skin slice | |
CN111954816A (en) | Method for detecting MAGEA4 | |
Garcia-Vasquez et al. | Development of a light microscopy stain for the sclerites of Gyrodactylus von Nordmann, 1832 (Monogenea) and related genera | |
CN110073760B (en) | Pre-wetting solution, staining solution and method for rapidly determining vitality of Iris tenuifolia seeds | |
Gill et al. | Exposure of nuclear antigens in formalin-fixed, paraffin-embedded necropsy human spinal cord tissue: detection of NeuN | |
DeJarnatt et al. | Glyoxal: a proposed substitute for formalin in H&E and special stains | |
CN113640520A (en) | Application of tissue transparency method and histology method in combination for detecting bacteria in tumor | |
Sutherland et al. | Probing cell wall structure and development by the use of antibodies: a personal perspective | |
De Neergaard et al. | Anatomy and histology of roots and root-soil boundary | |
CN106872243A (en) | RNA- protein-DNA original positions multiple staining the method for organization chip | |
Biswas et al. | SUMIC: a simple ultrafast multicolor immunolabelling and clearing approach for whole-organ and large tissue 3D imaging | |
CN105675880B (en) | Quickly detection low abundance proteins without closed immunoblotting | |
Watanabe et al. | Immunoelectron microscopic observation of chicken glucagon-like peptide (GLP)-1-containing cells in tissues derived from thin section, paraffin block and conventional method | |
Leathem et al. | Light microscopy: overview and basic methods | |
CN116380589B (en) | Pretreatment method and application of bone marrow tissue | |
CN107560917A (en) | A kind of processing method of miniature organ | |
JPH0368865A (en) | Inspection of pathological tissue and dehydrating agent therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160817 |