CN112525640A - Xylene-free paraffin embedding dehydration transparent agent - Google Patents
Xylene-free paraffin embedding dehydration transparent agent Download PDFInfo
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- CN112525640A CN112525640A CN202011397650.6A CN202011397650A CN112525640A CN 112525640 A CN112525640 A CN 112525640A CN 202011397650 A CN202011397650 A CN 202011397650A CN 112525640 A CN112525640 A CN 112525640A
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- 239000012188 paraffin wax Substances 0.000 title claims abstract description 17
- 238000006297 dehydration reaction Methods 0.000 title abstract description 31
- 230000018044 dehydration Effects 0.000 title abstract description 30
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 37
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 27
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 241000779819 Syncarpia glomulifera Species 0.000 claims abstract description 12
- 239000001739 pinus spp. Substances 0.000 claims abstract description 12
- 229940036248 turpentine Drugs 0.000 claims abstract description 12
- 239000008096 xylene Substances 0.000 claims abstract description 12
- 210000003734 kidney Anatomy 0.000 claims description 8
- 210000002784 stomach Anatomy 0.000 claims description 8
- 210000001198 duodenum Anatomy 0.000 claims description 7
- 210000004185 liver Anatomy 0.000 claims description 7
- 210000004556 brain Anatomy 0.000 claims description 5
- 239000000834 fixative Substances 0.000 claims 3
- 210000001519 tissue Anatomy 0.000 abstract description 66
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 38
- 238000000034 method Methods 0.000 abstract description 20
- 238000010186 staining Methods 0.000 abstract description 7
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 abstract description 6
- 210000000805 cytoplasm Anatomy 0.000 abstract description 4
- 230000006378 damage Effects 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 2
- 208000005156 Dehydration Diseases 0.000 description 26
- 235000019441 ethanol Nutrition 0.000 description 11
- 238000005406 washing Methods 0.000 description 9
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- 239000001293 FEMA 3089 Substances 0.000 description 4
- 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 4
- 239000001993 wax Substances 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000971 hippocampal effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241001289540 Fallopia convolvulus Species 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 244000050510 Cunninghamia lanceolata Species 0.000 description 1
- 241000729176 Fagopyrum dibotrys Species 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 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 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a xylene-free paraffin embedding dehydrating transparentizing agent, which is a mixture of n-butanol and turpentine as a dehydrating transparentizing agent in a paraffin embedding procedure to replace absolute ethyl alcohol and xylene, so that a processed tissue section is smooth, and the tissue is not brittle and hard; after HE staining, the nucleus and cytoplasm of the novel dehydrated tissue are clear, and the staining degree, color and transparency of the tissue are no worse than those of the tissue subjected to conventional dehydration; the invention adopts n-butyl alcohol and turpentine as the dehydration clearing agent, which can be used for tissue dehydration, can avoid the toxic effect of dimethylbenzene to human, and can reduce the damage of absolute alcohol over-dehydration to the tissue.
Description
Technical Field
The invention relates to a xylene-free paraffin embedding dehydration clearing agent.
Background
The quality of tissue embedding is the cornerstone of medical scientific research work, and the conventional embedding process comprises gradient ethanol dehydration, xylene transparence, wax immersion and the like. Wherein, the tissue dehydration process is a key link influencing the embedding quality.
The proper dehydration clearing agent can increase the rapidness of paraffin section preparation and ensure the quality of the section, which is of great importance for subsequent experiments such as tissue staining, immunohistochemistry and the like. However, the existing conventional dehydrating and clearing agent xylene has the defects of increasing the brittleness and hardening the texture of tissues, and the xylene volatilizes and can cause the toxic reaction of human bodies after being absorbed by the human bodies, so that a novel dehydrating and clearing agent with lower harm to the human bodies and better dehydrating and clearing degrees is urgently needed to be searched.
Disclosure of Invention
In view of the above, the invention provides a xylene-free paraffin embedding dehydration clearing agent, which can replace dehydration of absolute ethyl alcohol and clearing effect of xylene in a conventional paraffin embedding procedure. Tissue slices treated by the dehydrating and transparentizing agent are smooth, and tissues are not embrittled or hardened.
The invention provides a xylene-free paraffin embedded dehydrating transparentizing agent, which is prepared by mixing n-butyl alcohol and turpentine.
Preferably, the volume ratio of the n-butyl alcohol to the turpentine oil in the dehydrating and transparentizing agent is 1-7: 1-3, and more preferably, the volume ratio of the n-butyl alcohol to the turpentine oil in the dehydrating and transparentizing agent is 1: 1.
The dehydration clearing agent is used for dehydration in the process of paraffin embedding of isolated tissues.
In some embodiments, the ex vivo tissue employed in the present invention is brain, kidney, stomach, liver, duodenal tissue.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the mixture of normal butanol and turpentine as a dehydrating clearing agent in the paraffin embedding procedure to replace absolute ethyl alcohol and xylene, so that the processed tissue slices are smooth, and the tissues are not brittle and hard; after HE dyeing, the nucleus and cytoplasm of the tissue treated by the novel dehydration clearing agent are clear, and the staining degree, color and transparency of the tissue are no different from those of the tissue treated by the conventional dehydration procedure; the invention adopts n-butyl alcohol and turpentine oil to dehydrate tissues, which can not only avoid the toxic effect of dimethylbenzene to human, but also reduce the damage of anhydrous alcohol over dehydration to tissues.
Drawings
FIG. 1 shows HE staining results of isolated tissues after dehydration embedding is performed by using a conventional tissue embedding dehydration clearing agent;
FIG. 2 shows HE staining results of each isolated tissue after dehydrating and embedding by using the tissue embedding dehydrating clearing agent of the invention;
FIG. 3 shows the immunohistochemical staining results of each isolated tissue after dehydrating and embedding with the tissue embedding dehydrating clearing agent of the present invention;
FIG. 4 shows immunohistochemical staining of each isolated tissue after dehydrating and embedding with conventional tissue embedding dehydrating clearing agent;
in the figure, 1 is liver isolated tissue, 2 is hippocampal isolated tissue, 3 is cerebral cortex isolated tissue, 4 is kidney isolated tissue, 5 is stomach isolated tissue, and 6 is duodenum isolated tissue.
Detailed Description
The invention provides a xylene-free paraffin-embedded dehydrating transparentizing agent, which is prepared by mixing n-butyl alcohol and turpentine, wherein the volume ratio of the n-butyl alcohol to the turpentine in the dehydrating transparentizing agent is 1: 1. When the volume ratio of the n-butyl alcohol to the turpentine oil in the dehydrating and clearing agent is 1-7: 1-3, the dehydrating effect is close to the following experimental result.
The dehydration clearing agent is used for carrying out a tissue paraffin embedding test, and the specific test process is as follows:
1. materials and methods
1.1 Experimental reagents
Absolute ethyl alcohol (shin & shin science and technology development ltd., Tianjin, 20190728); xylene (tianjin standard technologies limited, 20171212); n-butanol (Fuchen chemical reagent factory 2011210, Tianjin); turpentine (OboKai chemical Co., Ltd., Tianjin, 190318); neutral gums (chinese shanghai brand model factory, 20161113); bax (Abcam corporation, ab 32503); NeUN (baaode bioengineering, ltd, cs 89330); general secondary antibody (bosch de bioengineering ltd, 14D12L24B 2722); DAB color development liquid (ZLI-9018, China fir Jinqiao biotechnology limited in Beijing)
1.2 methods
The multiple cerebral infarction model rat treated by the stomach-perfused polygonum convolvulus is killed by the decapitation method, the tissues of the brain, the liver, the kidney, the stomach and the duodenum are taken, the tissues are fixed by 4 percent paraformaldehyde for 48 hours, the running water is used for washing for 2 hours, and the tissues are trimmed to the size of 1 multiplied by 0.5cm on a wax plate. All tissues were subjected to a novel dehydration procedure: 60% ethanol 15min, 70% ethanol 15min, 80% ethanol 15min, 90% ethanol 15min, n-butanol at 60 ℃: turpentine (volume ratio 1: 1) for 3h, and embedding in paraffin at 60 deg.C for 30min and 60 deg.C for 30 min. A paraffin microtome was used to prepare 5 μm thick sections for HE staining and immunohistochemical staining, and the results were compared with those of tissues prepared by a conventional dehydration procedure. Conventional dehydration procedure is shown in table 1 below, and a novel dehydration procedure using the dehydrating transparentizing agent of the present invention is shown in table 2 below.
TABLE 1 conventional dehydration procedure
TABLE 2 novel dehydration procedure
HE staining process: xylene I, II for 20min each, 100% ethanol I, II for 10min each, 95%, 80%, 70% ethanol for 5min each, hematoxylin staining for 5min, tap water washing for 2min, hydrochloric acid-ethanol differentiation for 2s, 5% ammonia water rewet for 30s, 80%, 95% ethanol for 3min each, eosin staining for 2min, 80%, 95%, 100% ethanol for 5min each, xylene I, II for 10min each, neutral gum: turpentine (40: 1) was mixed, sealed, air dried, and observed under microscope. SABC immunohistochemical staining: dewaxing the slices by xylene I, II for 20min respectively in sequence, dehydrating by 100%, 95%, 80% and 70% ethanol for 5min respectively, washing by pure water for 3min, washing by PBS for 5min (repeated 3 times), washing by citric acid for 3min by 40 fire and antigen repairing by 20min by 20 fire by 2min, incubating by 3% H2O2 for 30min, washing by PBS for 5min (repeated 3 times), sealing by serum in an incubator at 37 ℃ for 20min, dropwise adding a Bax primary antibody (1: 200), standing overnight at 4 ℃, washing by PBS for 5min (repeated 3 times), incubating by a secondary antibody in the incubator at 37 ℃ for 30min, washing by PBS for 5min (repeated 3 times), incubating by SABC working solution at 37 ℃ for 30min, developing DAB, observing under a mirror, finishing developing color by pure water washing, counterstaining by hematoxylin 2min, dehydrating by gradient alcohol, and enabling the xylene: turpentine (40: 1) sealing sheet.
2. Results
2.1HE staining results
Comparing fig. 1 and fig. 2 (in the figures, serial numbers 1-6 are liver isolated tissue, hippocampal isolated tissue, cerebral cortex isolated tissue, kidney isolated tissue, stomach isolated tissue, and duodenum isolated tissue in sequence), it can be found that the paraffin section of the isolated tissue obtained by dehydrating and embedding the isolated tissue by using the dehydrating and clearing agent of the present invention is smooth, a complete wax band can be formed, and the cut wax sheet is very flat and has no phenomena of brittle tissue and hard texture.
After the in vitro tissue subjected to dehydration embedding treatment is subjected to HE dyeing, the cell nucleus is purple blue, the cytoplasm is red, the cell nucleus and the cytoplasm are clear, and the color, the coloring degree and the transparency of a tissue section are not obviously different from those of the tissue section subjected to conventional dehydration treatment. In HE staining, we observed that brain tissue cells have the appearance of nuclear pyknosis, neuron loss, slight liver steatosis and no obvious pathological changes of kidney, stomach and duodenum. When the tissue is subjected to He staining by conventional dehydration embedding treatment, the tissue patch is not tightly folded and curled, and part of the tissue is fractured and has incomplete structure such as intestinal tissue.
2.2 immunohistochemical results
The invention detects the expression of the apoptosis-promoting factor BAX in the brain, the kidney, the stomach, the liver and the duodenum of a multiple cerebral infarction model rat treated by polygonum convolvulus through immunohistochemical staining, and the result shows that the positive expression of brown yellow particles can be seen in each isolated tissue after dehydration embedding treatment by using the method, and the result is not different from the conventional dehydration procedure, as shown in figures 3-4 (in the figures, the serial numbers 1-6 are sequentially liver isolated tissue, hippocampal isolated tissue, brain cortex isolated tissue, kidney isolated tissue, stomach isolated tissue and duodenum isolated tissue).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. The paraffin embedded dehydrating clearing agent without xylene is characterized by being prepared by mixing n-butyl alcohol and turpentine.
2. The xylene-free, paraffin-embedded dehydrating transparentizer of claim 1, wherein the volume ratio of n-butanol to turpentine in the dehydrating transparentizer is 1-7: 1-3.
3. The xylene-free paraffin-embedded dehydrating fixative as claimed in claim 1 or 2, wherein the dehydrating fixative is used during paraffin embedding of ex vivo tissue.
4. The xylene-free paraffin-embedded dehydrating fixative as claimed in claim 3, wherein the ex vivo tissue is brain, kidney, stomach, liver, duodenum tissue.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011397650.6A CN112525640A (en) | 2020-12-04 | 2020-12-04 | Xylene-free paraffin embedding dehydration transparent agent |
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| CN202011397650.6A CN112525640A (en) | 2020-12-04 | 2020-12-04 | Xylene-free paraffin embedding dehydration transparent agent |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1068653A (en) * | 1991-08-26 | 1993-02-03 | 广西农学院 | Tranparency agent for biological sample slide |
| CN102279121A (en) * | 2011-06-28 | 2011-12-14 | 黑龙江八一农垦大学 | Green making technology of animal tissue paraffin section |
-
2020
- 2020-12-04 CN CN202011397650.6A patent/CN112525640A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1068653A (en) * | 1991-08-26 | 1993-02-03 | 广西农学院 | Tranparency agent for biological sample slide |
| CN102279121A (en) * | 2011-06-28 | 2011-12-14 | 黑龙江八一农垦大学 | Green making technology of animal tissue paraffin section |
Non-Patent Citations (5)
| Title |
|---|
| 付小一等: "911柔性生物制片透明脱蜡剂在石蜡制片中的应用", 《宜春学院学报》 * |
| 张天杰等: "正丁醇作透明剂在病理组织切片中的应用", 《湘南学院学报(医学版)》 * |
| 田珑: "正丁醇脱水制作肝组织石蜡切片", 《解剖科学进展》 * |
| 胥维勇: "3种环保透明剂在抗酸染色中的应用", 《诊断病理学杂志》 * |
| 骆利康: "介绍一种以正丁醇代替二甲苯的常规制片法", 《浙江医学》 * |
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