CN116380589B - Pretreatment method and application of bone marrow tissue - Google Patents
Pretreatment method and application of bone marrow tissue Download PDFInfo
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- CN116380589B CN116380589B CN202310370779.5A CN202310370779A CN116380589B CN 116380589 B CN116380589 B CN 116380589B CN 202310370779 A CN202310370779 A CN 202310370779A CN 116380589 B CN116380589 B CN 116380589B
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- 210000001519 tissue Anatomy 0.000 title claims abstract description 103
- 210000001185 bone marrow Anatomy 0.000 title claims abstract description 46
- 238000002203 pretreatment Methods 0.000 title claims abstract description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 75
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 238000002791 soaking Methods 0.000 claims abstract description 24
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 232
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 65
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 48
- 238000000034 method Methods 0.000 claims description 40
- 239000012188 paraffin wax Substances 0.000 claims description 38
- 238000006297 dehydration reaction Methods 0.000 claims description 33
- 230000018044 dehydration Effects 0.000 claims description 28
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 27
- 239000008096 xylene Substances 0.000 claims description 17
- 239000012153 distilled water Substances 0.000 claims description 13
- 238000011010 flushing procedure Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000007901 in situ hybridization Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000427 antigen Substances 0.000 abstract description 2
- 102000036639 antigens Human genes 0.000 abstract description 2
- 108091007433 antigens Proteins 0.000 abstract description 2
- 238000002509 fluorescent in situ hybridization Methods 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 92
- 238000005406 washing Methods 0.000 description 37
- 230000029087 digestion Effects 0.000 description 30
- 238000009396 hybridization Methods 0.000 description 25
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 23
- 238000010438 heat treatment Methods 0.000 description 20
- 238000004925 denaturation Methods 0.000 description 15
- 239000000523 sample Substances 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 12
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 10
- 239000004365 Protease Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 230000036425 denaturation Effects 0.000 description 10
- 238000001035 drying Methods 0.000 description 10
- 238000007789 sealing Methods 0.000 description 10
- 230000008439 repair process Effects 0.000 description 9
- 239000000203 mixture Substances 0.000 description 6
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 5
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- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
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- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
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- 206010025323 Lymphomas Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
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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
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Abstract
The invention provides a pretreatment method and application of bone marrow tissue; the pretreatment method realizes the complete naked decalcification of the first bone marrow tissue by soaking in EDTA decalcification solution, and performs the second decalcification of the surface of the bone marrow tissue by hydrochloric acid decalcification solution, so that not only can a good decalcification effect be realized, but also the section can not be influenced, the antigen is not destroyed, and the pretreated bone marrow tissue can be subjected to FISH detection, thereby effectively avoiding false negatives.
Description
Technical Field
The invention belongs to the technical field of biopsy tissue treatment, and particularly relates to a bone marrow tissue pretreatment method and application.
Background
Fluorescence In Situ Hybridization (FISH) is a detection technique that utilizes non-radioactive fluorescent signals to perform in situ hybridization on a sample. FISH can assist in diagnosing blood diseases with characteristic genetics, and clinical FISH probes related to blood tumor are close to 100, and about 60 are frequently used. The application range is increasing, and the application range comprises various blood tumors such as acute leukemia, chronic leukemia, myelodysplastic syndrome (MDS), multiple Myeloma (MM), lymphoma and the like. The FISH test samples are widely available and include blood, bone marrow fluid, tissue, exfoliated cells, amniotic fluid, ascites, etc. Since a fixed decalcification pretreatment is required before tissue sections of bone marrow biopsies, and decalcification damages DNA, thereby affecting the result of FISH detection, false negatives appear, so that bone marrow tissues subjected to general decalcification pretreatment are not suitable for FISH detection.
Disclosure of Invention
The invention provides a pretreatment method and application of bone marrow tissue, which are used for solving the defect that the prior art bone marrow tissue decalcification pretreatment damages DNA and causes false negative.
Based on the above, the technical scheme of the invention is as follows:
the first object of the present invention is to provide a bone marrow tissue pretreatment method, comprising the following steps:
s1, placing the puncture tissue into a fixing solution for fixing for 16-24 hours, and flushing with flowing water for the first time;
s2, continuously decalcifying the tissue by using EDTA decalcification solution for 7-8h, and flushing the tissue by using running water for the second time;
s3, carrying out gradient dehydration and paraffin embedding on the tissue, and then roughly repairing;
s4, soaking the embedded tissues in a hydrochloric acid solution containing formaldehyde for 15-30min.
In one embodiment, the formaldehyde-containing hydrochloric acid solution is prepared from 10% hydrochloric acid, 5% formaldehyde and 85% distilled water by volume.
In one embodiment, the EDTA decalcification solution has a concentration of 0.5mol/L and a pH of 8.0.
In one embodiment, the fixative solution described in step S1 is used to preserve the original morphological structure of cells and tissues, including but not limited to Bouin' S fixative solution or formaldehyde fixative solution.
In one embodiment, the gradient dewatering step in step S3 is: 10% formalin is soaked for 1h, then soaked for 1h by 75% alcohol, soaked for 1h by 95% alcohol and repeated for 2 times, soaked for 1h by absolute alcohol and repeated for 3 times, soaked for 40min by xylene and repeated for 3 times, soaked for 1h by paraffin and repeated for 3 times.
A second object of the present invention is to propose the use of the reagents in the pretreatment method described above for the preparation of a bone marrow tissue pretreatment kit.
A third object of the present invention is to provide a method for preparing paraffin sections of bone marrow tissue, comprising the above-mentioned bone marrow tissue pretreatment method.
In one embodiment, the method of making bone marrow tissue paraffin sections further comprises the step of slicing the pretreated tissue.
A fourth object of the present invention is to provide paraffin sections obtained by the above-mentioned method for producing bone marrow tissue paraffin sections.
A fifth object of the present invention is to propose the use of said paraffin sections for preparing fluorescent in situ hybridization detection tissue samples.
Compared with the prior art, the invention has the beneficial effects that:
the pretreatment method of the invention realizes the complete naked decalcification of the bone marrow tissue by soaking in the EDTA decalcification solution, and carries out the decalcification of the surface of the bone marrow tissue for the second time by using the hydrochloric acid decalcification solution, thereby having high decalcification efficiency and being capable of realizing good decalcification effect; the method has the advantages that the section is not affected, the antigen is not destroyed, the pretreated bone marrow tissue can be subjected to FISH detection, and false negative can be effectively avoided.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of HE staining and FISH detection signals of tissue sections according to example 1 of the present invention.
FIG. 2 shows the results of HE staining and FISH detection signals performed on tissue sections according to example 2 of the present invention.
FIG. 3 shows the results of HE staining and FISH detection signals from tissue sections according to example 3 of the present invention.
FIG. 4 shows the results of HE staining and FISH detection signals from tissue sections according to example 3 of the present invention.
FIG. 5 shows the results of HE staining and FISH detection signals from tissue sections according to example 3 of the present invention.
FIG. 6 shows the results of HE staining and FISH detection signals of the tissue sections of comparative example 1 of the present invention.
FIG. 7 shows the results of HE staining and FISH detection signals of the tissue sections of comparative example 2 according to the present invention.
FIG. 8 shows the results of HE staining and FISH detection signals of the tissue sections of comparative example 3 according to the present invention.
FIG. 9 shows the results of HE staining and FISH detection signals of the tissue sections of comparative example 4 according to the present invention.
FIG. 10 shows the results of HE staining and FISH detection signals of the tissue sections of comparative example 5 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The decalcification solution used in the following examples was composed as follows:
EDTA decalcification solution composition: the concentration was 0.5mol/L, and the pH was adjusted to 8.0 with sodium hydroxide.
Hydrochloric acid decalcification liquid comprises the following components: 100ml of hydrochloric acid (commercially available, content 36-38%) plus 50ml of formaldehyde plus 850ml of distilled water.
The means of operation used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
1.1 bone marrow tissue pretreatment method:
bone marrow tissue fixation-decalcification-dehydration-embedding: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 20 hours, sampling, washing with flowing water for 20 minutes, decalcification with EDTA decalcification solution for 7 hours (complete decalcification), washing with flowing water for 15 minutes, gradient dehydration, paraffin embedding, rough repair, soaking with hydrochloric acid decalcification solution for 20 minutes (surface decalcification), and slicing.
The gradient dehydration procedure was: 10% formalin 1h, 75% alcohol 1h,95% alcohol 1h x 2, absolute alcohol 1h x 3, xylene 40min x 3, paraffin 1h x 3.
1.2HE staining procedure: baking slices at 80 ℃ for half an hour after 3 mu m slicing, 10min for 3 of a dewaxing agent, 3min for 3 of absolute ethyl alcohol, 3min for 2 of 95% ethyl alcohol, 3min for 75% ethyl alcohol, 2min for running water washing, 6min for hematoxylin, 2min for running water washing, 3-5 s for differentiation of 4% hydrochloric acid alcohol, 2min for running water washing, 10s for returning 1% ammonia water, 2min for running water washing, 3-5 s for eosin, running water washing, gradient alcohol dehydration, blow drying and sealing the slices, and waiting to be detected.
1.3 pretreatment of FISH detection slides: slice dewaxing-pretreatment of slides-heating pretreatment-protease digestion treatment-gradient dehydration.
(1) Slide dewaxing (slide must not exceed 5): the 3 μm slide was placed in an incubator at 65℃for 4 hours, and the following steps were performed at room temperature, xylene (10 min) →xylene (10 min) →100% ethanol (5 min).
(2) Pretreatment of the slide: 100% ethanol (2 min) → 85% ethanol (2 min) → 70% ethanol (2 min) → distilled water 3min.
(3) And (3) heating pretreatment: distilled water is placed in a water bath kettle to be heated, the temperature is 90 ℃ and the heating time is 30min, and the heating time is required to be 1h in advance. Cooled to room temperature and washed 2 times with 2 XSSC for 5min each.
(4) Protease digestion treatment: the pepsin solution (10 mM HCl) of 6mg/mL is digested, the digestion time depends on the thickness of the lamina and the tissue, the thickness of the lamina is preferably 2-3 μm, the digestion time of the tissue is about 20min, the digestion time of the lymphoid tissue is about 40min, and the incubation temperature is 37 ℃. After digestion is complete, the cells are washed 2 times with 2 XSSC for 5min each.
(5) Gradient dehydration: 70% ethanol (1 min) → 90% ethanol (1 min) → 100% ethanol (1 min), and naturally drying for 15min.
Probe procedure: probe-denaturation hybridization-dehydration after hybridization.
(1) And (3) upper probe: mu.L of probe-use liquid, a cover slip and a sealing gel are dripped into a designated area.
(2) Denaturation hybridization: thermoBrite system procedure is: denaturation: 83 ℃ for 5min; hybridization: 42℃overnight. Note that: before cover hybridization, ensure humidity card saturation.
(3) Post hybridization treatment
a. Slide (slide must not exceed 5): the slides were rinsed in 0.3% NP-40/0.4 XSSC at 67℃for 1.5min. Then, the mixture was left at room temperature for 0.1% NP-40/2 XSSC, and taken out after 0.5 min.
b. Dehydrating: and (3) 70% ethanol (1 min), 90% ethanol (1 min), 100% ethanol (1 min) and naturally drying for 20min.
C. And (3) cover plate: add 5. Mu.L DAPI and coverslip.
Signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
1.4 results:
as shown in FIG. 1, the tissue structure of the section after HE staining shows clear, clear nucleus contrast and clean background. FISH detects the visible signal and the signal is sufficient to analyze the result.
1.5 experimental conclusion: the EDTA decalcification solution for bone marrow tissue pretreatment is used for decalcification for 7 hours to completely decalcifize, and paraffin is embedded and then soaked in the hydrochloric acid decalcification solution for 20 minutes to realize surface decalcification. This combined decalcification method allows for better HE staining of bone marrow tissue sections and for FISH detection.
Example 2
1.1 bone marrow tissue pretreatment method:
bone marrow tissue fixation-decalcification-dehydration-embedding: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 16 hours, sampling, washing with flowing water for 15 minutes, decalcification with EDTA decalcification solution for 8 hours (complete decalcification), washing with flowing water for 20 minutes, gradient dehydration, paraffin embedding, rough repair, soaking with hydrochloric acid decalcification solution for 15 minutes (surface decalcification), and slicing.
The gradient dehydration procedure was: 10% formalin 1h, 75% alcohol 1h,95% alcohol 1h x 2, absolute alcohol 1h x 3, xylene 40min x 3, paraffin 1h x 3.
1.2HE staining procedure: baking slices at 80 ℃ for half an hour after 3 mu m slicing, 10min for 3 of a dewaxing agent, 3min for 3 of absolute ethyl alcohol, 3min for 2 of 95% ethyl alcohol, 3min for 75% ethyl alcohol, 2min for running water washing, 6min for hematoxylin, 2min for running water washing, 3-5 s for differentiation of 4% hydrochloric acid alcohol, 2min for running water washing, 10s for returning 1% ammonia water, 2min for running water washing, 3-5 s for eosin, running water washing, gradient alcohol dehydration, blow drying and sealing the slices, and waiting to be detected.
1.3 pretreatment of FISH detection slides: slice dewaxing-pretreatment of slides-heating pretreatment-protease digestion treatment-gradient dehydration.
(1) Slide dewaxing (slide must not exceed 5): the 3 μm slide was placed in an incubator at 65℃for 4 hours, and the following steps were performed at room temperature, xylene (10 min) →xylene (10 min) →100% ethanol (5 min).
(2) Pretreatment of the slide: 100% ethanol (2 min) → 85% ethanol (2 min) → 70% ethanol (2 min) → distilled water 3min.
(3) And (3) heating pretreatment: distilled water is placed in a water bath kettle to be heated, the temperature is 90 ℃ and the heating time is 30min, and the heating time is required to be 1h in advance. Cooled to room temperature and washed 2 times with 2 XSSC for 5min each.
(4) Protease digestion treatment: the pepsin solution (10 mM HCl) of 6mg/mL is digested, the digestion time depends on the thickness of the lamina and the tissue, the thickness of the lamina is preferably 2-3 μm, the digestion time of the tissue is about 20min, the digestion time of the lymphoid tissue is about 40min, and the incubation temperature is 37 ℃. After digestion is complete, the cells are washed 2 times with 2 XSSC for 5min each.
(5) Gradient dehydration: 70% ethanol (1 min) → 90% ethanol (1 min) → 100% ethanol (1 min), and naturally drying for 20min.
Probe procedure: probe-denaturation hybridization-dehydration after hybridization.
(1) And (3) upper probe: mu.L of probe-use liquid, a cover slip and a sealing gel are dripped into a designated area.
(2) Denaturation hybridization: thermoBrite system procedure is: denaturation: 83 ℃ for 5min; hybridization: 42℃overnight. Note that: before cover hybridization, ensure humidity card saturation.
(3) Post hybridization treatment
a. Slide (slide must not exceed 5): the slides were rinsed in 0.3% NP-40/0.4 XSSC at 67℃for 1.5min. Then, the mixture was left at room temperature for 0.1% NP-40/2 XSSC, and taken out after 0.5 min.
b. Dehydrating: 70% ethanol (1 min) → 90% ethanol (1 min) → 100% ethanol (1 min), and naturally drying for 15min.
C. And (3) cover plate: add 5. Mu.L DAPI and coverslip.
Signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
1.4 results: as shown in FIG. 2, the tissue structure of the section after HE staining shows clear, clear nucleus contrast and clean background. FISH detects the visible signal and the signal is sufficient to analyze the result.
1.5 experimental conclusion: the EDTA decalcification solution for bone marrow tissue pretreatment is used for decalcification for 8 hours to completely decalcifize, and paraffin is embedded and then soaked in the hydrochloric acid decalcification solution for 15 minutes to realize surface decalcification. This combined decalcification method allows for better HE staining of bone marrow tissue sections and for FISH detection.
Example 3
1.1 bone marrow tissue pretreatment method:
bone marrow tissue fixation-decalcification-dehydration-embedding: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 24 hours, sampling, flushing with flowing water for 20 minutes, decalcification with EDTA decalcification solution for 7 hours (complete decalcification), flushing with flowing water for 20 minutes, gradient dehydration, paraffin embedding, rough repair, soaking in hydrochloric acid decalcification solution for 15 minutes (surface decalcification), and slicing.
The gradient dehydration procedure was: 10% formalin 1h, 75% alcohol 1h,95% alcohol 1h x 2, absolute alcohol 1h x 3, xylene 40min x 3, paraffin 1h x 3.
1.2HE staining procedure: baking slices at 80 ℃ for half an hour after 3 mu m slicing, 10min for 3 of a dewaxing agent, 3min for 3 of absolute ethyl alcohol, 3min for 2 of 95% ethyl alcohol, 3min for 75% ethyl alcohol, 2min for running water washing, 6min for hematoxylin, 2min for running water washing, 3-5 s for differentiation of 4% hydrochloric acid alcohol, 2min for running water washing, 10s for returning 1% ammonia water, 2min for running water washing, 3-5 s for eosin, running water washing, gradient alcohol dehydration, blow drying and sealing the slices, and waiting to be detected.
1.3 pretreatment of FISH detection slides: slice dewaxing-pretreatment of slides-heating pretreatment-protease digestion treatment-gradient dehydration.
(1) Slide dewaxing (slide must not exceed 5): the 3 μm slide was placed in an incubator at 65℃for 4 hours, and the following steps were performed at room temperature, xylene (10 min) →xylene (10 min) →100% ethanol (5 min).
(2) Pretreatment of the slide: 100% ethanol (2 min) → 85% ethanol (2 min) → 70% ethanol (2 min) → distilled water 3min.
(3) And (3) heating pretreatment: distilled water is placed in a water bath kettle to be heated, the temperature is 90 ℃ and the heating time is 30min, and the heating time is required to be 1h in advance. Cooled to room temperature and washed 2 times with 2 XSSC for 5min each.
(4) Protease digestion treatment: the pepsin solution (10 mM HCl) of 6mg/mL is digested, the digestion time depends on the thickness of the lamina and the tissue, the thickness of the lamina is preferably 2-3 μm, the digestion time of the tissue is about 20min, the digestion time of the lymphoid tissue is about 40min, and the incubation temperature is 37 ℃. After digestion is complete, the cells are washed 2 times with 2 XSSC for 5min each.
(5) Gradient dehydration: 70% ethanol (1 min) → 90% ethanol (1 min) → 100% ethanol (1 min), and naturally drying for 15min.
Probe procedure: probe-denaturation hybridization-dehydration after hybridization.
(1) And (3) upper probe: mu.L of probe-use liquid, a cover slip and a sealing gel are dripped into a designated area.
(2) Denaturation hybridization: thermoBrite system procedure is: denaturation: 83 ℃ for 5min; hybridization: 42℃overnight. Note that: before cover hybridization, ensure humidity card saturation.
(3) Post hybridization treatment
a. Slide (slide must not exceed 5): the slides were rinsed in 0.3% NP-40/0.4 XSSC at 67℃for 1.5min. Then, the mixture was left at room temperature for 0.1% NP-40/2 XSSC, and taken out after 0.5 min.
b. Dehydrating: and (3) 70% ethanol (1 min), 90% ethanol (1 min), 100% ethanol (1 min), and naturally drying for at least 10min.
C. And (3) cover plate: add 5. Mu.L DAPI and coverslip.
Signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
1.4 results: as shown in FIG. 3, the tissue structure of the section after HE staining shows clear, clear nucleus contrast and clean background. FISH detects the visible signal and the signal is sufficient to analyze the result.
1.5 experimental conclusion: the EDTA decalcification solution for bone marrow tissue pretreatment is used for decalcification for 7 hours to completely decalcifize, and paraffin is embedded and then soaked in the hydrochloric acid decalcification solution for 15 minutes to realize surface decalcification. This combined decalcification method allows for better HE staining of bone marrow tissue sections and for FISH detection.
Example 4
1.1 bone marrow tissue pretreatment method:
bone marrow tissue fixation-decalcification-dehydration-embedding: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 18 hours, sampling, washing with flowing water for 20 minutes, decalcification with EDTA decalcification solution for 8 hours (complete decalcification), washing with flowing water for 15 minutes, gradient dehydration, paraffin embedding, rough repair, soaking in hydrochloric acid decalcification solution for 18 minutes (surface decalcification), and slicing.
The gradient dehydration procedure was: 10% formalin 1h, 75% alcohol 1h,95% alcohol 1h x 2, absolute alcohol 1h x 3, xylene 40min x 3, paraffin 1h x 3.
1.2HE staining procedure: baking slices at 80 ℃ for half an hour after 3 mu m slicing, 10min for 3 of a dewaxing agent, 3min for 3 of absolute ethyl alcohol, 3min for 2 of 95% ethyl alcohol, 3min for 75% ethyl alcohol, 2min for running water washing, 6min for hematoxylin, 2min for running water washing, 3-5 s for differentiation of 4% hydrochloric acid alcohol, 2min for running water washing, 10s for returning 1% ammonia water, 2min for running water washing, 3-5 s for eosin, running water washing, gradient alcohol dehydration, blow drying and sealing the slices, and waiting to be detected.
1.3 pretreatment of FISH detection slides: slice dewaxing-pretreatment of slides-heating pretreatment-protease digestion treatment-gradient dehydration.
(1) Slide dewaxing (slide must not exceed 5): the 3 μm slide was placed in an incubator at 65℃for 4 hours, and the following steps were performed at room temperature, xylene (10 min) →xylene (10 min) →100% ethanol (5 min).
(2) Pretreatment of the slide: 100% ethanol (2 min) → 85% ethanol (2 min) → 70% ethanol (2 min) → distilled water 3min.
(3) And (3) heating pretreatment: distilled water is placed in a water bath kettle to be heated, the temperature is 90 ℃ and the heating time is 30min, and the heating time is required to be 1h in advance. Cooled to room temperature and washed 2 times with 2 XSSC for 5min each.
(4) Protease digestion treatment: the pepsin solution (10 mM HCl) of 6mg/mL is digested, the digestion time depends on the thickness of the lamina and the tissue, the thickness of the lamina is preferably 2-3 μm, the digestion time of the tissue is about 20min, the digestion time of the lymphoid tissue is about 40min, and the incubation temperature is 37 ℃. After digestion is complete, the cells are washed 2 times with 2 XSSC for 5min each.
(5) Gradient dehydration: 70% ethanol (1 min) → 90% ethanol (1 min) → 100% ethanol (1 min), and naturally drying for 15min.
Probe procedure: probe-denaturation hybridization-dehydration after hybridization.
(1) And (3) upper probe: mu.L of probe-use liquid, a cover slip and a sealing gel are dripped into a designated area.
(2) Denaturation hybridization: thermoBrite system procedure is: denaturation: 83 ℃ for 5min; hybridization: 42℃overnight. Note that: before cover hybridization, ensure humidity card saturation.
(3) Post hybridization treatment
a. Slide (slide must not exceed 5): the slides were rinsed in 0.3% NP-40/0.4 XSSC at 67℃for 1.5min. Then, the mixture was left at room temperature for 0.1% NP-40/2 XSSC, and taken out after 0.5 min.
b. Dehydrating: and (3) 70% ethanol (1 min), 90% ethanol (1 min), 100% ethanol (1 min), and naturally drying for at least 10min.
C. And (3) cover plate: add 5. Mu.L DAPI and coverslip.
Signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
1.4 results: as shown in FIG. 4, the tissue structure of the section after HE staining showed clear, clear nucleus contrast and clean background. FISH detects the visible signal and the signal is sufficient to analyze the result.
1.5 experimental conclusion: the EDTA decalcification solution for bone marrow tissue pretreatment is used for decalcification for 8 hours to completely decalcifize, and paraffin is embedded and then soaked in the hydrochloric acid decalcification solution for 18 minutes to realize surface decalcification. This combined decalcification method allows for better HE staining of bone marrow tissue sections and for FISH detection.
Example 5
1.1 bone marrow tissue pretreatment method:
bone marrow tissue fixation-decalcification-dehydration-embedding: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 18 hours, sampling, washing for 15 minutes with running water, decalcification with EDTA decalcification solution for 7 hours (complete decalcification), washing for 20 minutes with running water, gradient dehydration, paraffin embedding, rough repair, soaking in hydrochloric acid decalcification solution for 20 minutes (surface decalcification), and slicing.
The gradient dehydration procedure was: 10% formalin 1h, 75% alcohol 1h,95% alcohol 1h x 2, absolute alcohol 1h x 3, xylene 40min x 3, paraffin 1h x 3.
1.2HE staining procedure: baking slices at 80 ℃ for half an hour after 3 mu m slicing, 10min for 3 of a dewaxing agent, 3min for 3 of absolute ethyl alcohol, 3min for 2 of 95% ethyl alcohol, 3min for 75% ethyl alcohol, 2min for running water washing, 6min for hematoxylin, 2min for running water washing, 3-5 s for differentiation of 4% hydrochloric acid alcohol, 2min for running water washing, 10s for returning 1% ammonia water, 2min for running water washing, 3-5 s for eosin, running water washing, gradient alcohol dehydration, blow drying and sealing the slices, and waiting to be detected.
1.3 pretreatment of FISH detection slides: slice dewaxing-pretreatment of slides-heating pretreatment-protease digestion treatment-gradient dehydration.
(1) Slide dewaxing (slide must not exceed 5): the 3 μm slide was placed in an incubator at 65℃for 4 hours, and the following steps were performed at room temperature, xylene (10 min) →xylene (10 min) →100% ethanol (5 min).
(2) Pretreatment of the slide: 100% ethanol (2 min) → 85% ethanol (2 min) → 70% ethanol (2 min) → distilled water 3min.
(3) And (3) heating pretreatment: distilled water is placed in a water bath kettle to be heated, the temperature is 90 ℃ and the heating time is 30min, and the heating time is required to be 1h in advance. Cooled to room temperature and washed 2 times with 2 XSSC for 5min each.
(4) Protease digestion treatment: the pepsin solution (10 mM HCl) of 6mg/mL is digested, the digestion time depends on the thickness of the lamina and the tissue, the thickness of the lamina is preferably 2-3 μm, the digestion time of the tissue is about 20min, the digestion time of the lymphoid tissue is about 40min, and the incubation temperature is 37 ℃. After digestion is complete, the cells are washed 2 times with 2 XSSC for 5min each.
(5) Gradient dehydration: 70% ethanol (1 min) → 90% ethanol (1 min) → 100% ethanol (1 min), and naturally drying for 15min.
Probe procedure: probe-denaturation hybridization-dehydration after hybridization.
(1) And (3) upper probe: mu.L of probe-use liquid, a cover slip and a sealing gel are dripped into a designated area.
(2) Denaturation hybridization: thermoBrite system procedure is: denaturation: 83 ℃ for 5min; hybridization: 42℃overnight. Note that: before cover hybridization, ensure humidity card saturation.
(3) Post hybridization treatment
a. Slide (slide must not exceed 5): the slides were rinsed in 0.3% NP-40/0.4 XSSC at 67℃for 1.5min. Then, the mixture was left at room temperature for 0.1% NP-40/2 XSSC, and taken out after 0.5 min.
b. Dehydrating: and (3) 70% ethanol (1 min), 90% ethanol (1 min), 100% ethanol (1 min) and naturally drying for 20min.
C. And (3) cover plate: add 5. Mu.L DAPI and coverslip.
Signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
1.4 results:
as shown in FIG. 5, the tissue structure of the section after HE staining showed clear, clear nucleus contrast and clean background. FISH detects the visible signal and the signal is sufficient to analyze the result.
1.5 experimental conclusion: the EDTA decalcification solution for bone marrow tissue pretreatment is used for decalcification for 7 hours to completely decalcifize, and paraffin is embedded and then soaked in the hydrochloric acid decalcification solution for 20 minutes to realize surface decalcification. This combined decalcification method allows for better HE staining of bone marrow tissue sections and for FISH detection.
Comparative example 1
Conventional laboratory bone marrow tissue pretreatment method:
1.1 immobilization-decalcification gradient dehydration-embedding procedure is as follows: fixing a specimen by using a Bouin's fixing solution for 20 hours, treating the specimen by using a hydrochloric acid decalcification solution for 60 minutes, 10% formalin for 1 hour, 75% alcohol for 1 hour, 95% alcohol for 1 hour for 2 hours, absolute alcohol for 1 hour for 3 hours, dimethylbenzene for 40 minutes for 3 hours, paraffin for 1 hour for 3 hours, embedding paraffin, roughly repairing, soaking in the hydrochloric acid decalcification solution for 30 minutes, and slicing.
The other steps are the same as in example 1.
1.2 signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
As shown in FIG. 6, the tissue structure of the HE-stained section showed clear, clear nucleus contrast and clean background. FISH detection: no signal or very weak signal.
1.3 conclusion of experiment: the above conventional decalcification method HE stains well but cannot be used for FISH detection.
Comparative example 2
1.1 immobilization-decalcification gradient dehydration-embedding procedure is as follows: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 18 hours, drawing materials, washing for 15 minutes with running water, soaking for 60 minutes in hydrochloric acid decalcification solution, washing for 20 minutes with running water, 10% formalin for 1 hour, 75% alcohol for 1 hour, 95% alcohol for 1 hour for 2 hours, absolute alcohol for 1 hour for 3 hours, dimethylbenzene for 40 minutes for 3 hours, paraffin for 1 hour for 3 hours, paraffin embedding, rough repair, soaking for 20 minutes in hydrochloric acid decalcification solution, and slicing.
The other steps are the same as in example 2.
1.2 signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
Results: HE staining as shown in fig. 7: the tissue structure of the slice is clear, the nucleus is relatively clear, and the background is clean. FISH detection: no signal or very weak signal.
1.3 conclusion of experiment: the decalcification method HE above stains well but cannot be used for FISH detection.
Comparative example 3
1.1 immobilization-decalcification gradient dehydration-embedding procedure is as follows: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 24 hours, drawing materials, flushing with flowing water for 20 minutes, soaking in hydrochloric acid decalcification solution for 60 minutes, flushing with flowing water for 20 minutes, 10% formalin for 1 hour, 75% alcohol for 1 hour, 95% alcohol for 1 hour for 2 hours, absolute alcohol for 1 hour for 3 hours, dimethylbenzene for 40 minutes for 3 hours, paraffin for 1 hour for 3 hours, paraffin embedding for rough repair, soaking in hydrochloric acid decalcification solution for 15 minutes, and slicing.
The other steps are the same as in example 3.
1.2 signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
Results: HE staining as shown in fig. 8: the tissue structure of the slice is clear, the nucleus is relatively clear, and the background is clean. FISH detection: no signal or very weak signal.
1.3 conclusion of experiment: the above conventional decalcification method HE stains well but cannot be used for FISH detection.
Comparative example 4
1.1 immobilization-decalcification gradient dehydration-embedding procedure is as follows: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 18 hours, drawing materials, flushing with running water for 20 minutes, soaking in hydrochloric acid decalcification solution for 60 minutes, flushing with running water for 15 minutes, 10% formalin for 1 hour, 75% alcohol for 1 hour, 95% alcohol for 1 hour for 2 hours, absolute alcohol for 1 hour for 3 hours, dimethylbenzene for 40 minutes for 3 hours, paraffin for 1 hour for 3 hours, paraffin embedding for rough repair, soaking in hydrochloric acid decalcification solution for 60 minutes, and slicing.
The other steps are the same as in example 4.
1.2 signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
As shown in FIG. 9, the tissue structure of the HE-stained section showed clear, clear nucleus contrast and clean background. FISH detection: no signal or very weak signal.
1.3 conclusion of experiment: the decalcification method HE above stains well but cannot be used for FISH detection.
Comparative example 5
1.1 immobilization-decalcification gradient dehydration-embedding procedure is as follows: immediately after puncture, the puncture tissue is placed into a Bouin's fixing solution for fixing for 24 hours, drawing materials, washing for 15 minutes with running water, soaking in hydrochloric acid decalcification solution for 60 minutes, washing for 20 minutes with running water, 10% formalin for 1 hour, 75% alcohol for 1 hour, 95% alcohol for 1 hour for 2 hours, absolute alcohol for 1 hour for 3 hours, dimethylbenzene for 40 minutes for 3 hours, paraffin for 1 hour for 3 hours, paraffin embedding, rough repair, soaking in hydrochloric acid decalcification solution for 60 minutes, and slicing.
The other steps are the same as in example 5.
1.2 signal analysis: the color signal after the reaction was analyzed using an olympus BX53TRF fluorescence microscope.
As shown in FIG. 10, the tissue structure of the HE-stained section showed clear, clear nucleus contrast and clean background. FISH detection: no signal or very weak signal.
1.3 conclusion of experiment: the decalcification method HE above stains well but cannot be used for FISH detection.
Experimental example
Decalcification was performed using the same puncture tissue sample using the pretreatment methods provided in examples 1 to 5 and comparative examples 1 to 5, and tissue sections were prepared, and all sections were observed under a microscope. The decalcification solutions used in examples 1-5 and comparative examples 1-5 were evaluated for satisfaction by five pathologists together. The evaluation indexes comprise the slice integrity rate, the HE dyeing definition rate, the qualification rate of the FISH detection signal intensity (the stronger fluorescent signal is regarded as qualification) and the like. The satisfaction effect is satisfied by five pathologists in a consistent manner, and is unsatisfactory by more than one pathologist who is not satisfied. The evaluation results are shown in Table 1.
Table 1:
as can be readily seen from Table 1, the decalcification methods HE staining and FISH detection signals employed in examples 1-5 are significantly improved, fully illustrating the advantages over single decalcification solutions or conventional pretreatment methods.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (5)
1. A method for pretreating bone marrow tissue, comprising the steps of:
s1, placing a puncture tissue into Bouin's stationary liquid for fixing for 16-24 hours, and flushing with flowing water for the first time;
s2, continuously decalcifying the tissue by using EDTA decalcification solution for 7-8h, and flushing the tissue by using running water for the second time; the concentration of the EDTA decalcification solution is 0.5mol/L, and the PH is 8.0;
s3, carrying out gradient dehydration and paraffin embedding on the tissue, and then roughly repairing; the gradient dehydration step is as follows: soaking 10% formalin for 1h, sequentially soaking in 75% alcohol for 1h, soaking in 95% alcohol for 1h, repeating for 2 times, soaking in anhydrous alcohol for 1h, repeating for 3 times, soaking in xylene for 40min, repeating for 3 times, soaking in paraffin for 1h, repeating for 3 times;
s4, soaking the embedded tissue in a hydrochloric acid solution containing formaldehyde for 15-20min; the formaldehyde-containing hydrochloric acid solution is prepared from 10% of hydrochloric acid with the content of 35-38%, 5% of formaldehyde and 85% of distilled water according to the volume ratio.
2. A method for producing paraffin sections of bone marrow tissue, comprising the bone marrow tissue pretreatment method according to claim 1.
3. The method of claim 2, further comprising the step of slicing the pretreated tissue.
4. A paraffin section obtained by the method for producing a paraffin section for bone marrow tissue according to claim 2 or 3.
5. Use of paraffin sections according to claim 4 for the preparation of fluorescence in situ hybridization detection tissue samples.
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