CN107907397A - Collagenous fibres horse pine trichrome stain kit and preparation method thereof and colouring method - Google Patents
Collagenous fibres horse pine trichrome stain kit and preparation method thereof and colouring method Download PDFInfo
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- CN107907397A CN107907397A CN201711180864.6A CN201711180864A CN107907397A CN 107907397 A CN107907397 A CN 107907397A CN 201711180864 A CN201711180864 A CN 201711180864A CN 107907397 A CN107907397 A CN 107907397A
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- 235000008331 Pinus X rigitaeda Nutrition 0.000 title claims abstract description 19
- 235000011613 Pinus brutia Nutrition 0.000 title claims abstract description 19
- 241000018646 Pinus brutia Species 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000004040 coloring Methods 0.000 title claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 74
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000004043 dyeing Methods 0.000 claims abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 25
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims abstract description 16
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 claims abstract description 16
- RZUBARUFLYGOGC-MTHOTQAESA-L acid fuchsin Chemical compound [Na+].[Na+].[O-]S(=O)(=O)C1=C(N)C(C)=CC(C(=C\2C=C(C(=[NH2+])C=C/2)S([O-])(=O)=O)\C=2C=C(C(N)=CC=2)S([O-])(=O)=O)=C1 RZUBARUFLYGOGC-MTHOTQAESA-L 0.000 claims abstract description 14
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 claims abstract description 12
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000000975 dye Substances 0.000 claims description 16
- 239000012153 distilled water Substances 0.000 claims description 15
- 238000005374 membrane filtration Methods 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 11
- 235000019441 ethanol Nutrition 0.000 claims description 10
- 230000004069 differentiation Effects 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- AMWVZPDSWLOFKA-UHFFFAOYSA-N phosphanylidynemolybdenum Chemical compound [Mo]#P AMWVZPDSWLOFKA-UHFFFAOYSA-N 0.000 claims description 3
- 235000015281 sodium iodate Nutrition 0.000 claims description 3
- 229910052808 lithium carbonate Inorganic materials 0.000 claims description 2
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 claims description 2
- 229940032753 sodium iodate Drugs 0.000 claims description 2
- 239000011697 sodium iodate Substances 0.000 claims description 2
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims 1
- 238000004806 packaging method and process Methods 0.000 abstract description 5
- 238000010186 staining Methods 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 13
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 5
- 239000012128 staining reagent Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 230000036285 pathological change Effects 0.000 description 3
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- 241000754688 Cercaria Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000004411 aluminium Substances 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000984 vat dye Substances 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 108010017480 Hemosiderin Proteins 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 241000565675 Oncomelania Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000031816 Pathologic Dilatation Diseases 0.000 description 1
- 241000242677 Schistosoma japonicum Species 0.000 description 1
- 241001442514 Schistosomatidae Species 0.000 description 1
- WTCBONOLBHEDIL-UHFFFAOYSA-M Sodium iodate Chemical class [Na+].[O-]I(=O)=O WTCBONOLBHEDIL-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 235000015177 dried meat Nutrition 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000003786 sclera Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 206010040872 skin infection Diseases 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- -1 sweet ammonia Acid Chemical class 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000004048 vat dyeing Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
-
- 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
- G01N1/31—Apparatus therefor
-
- 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
- G01N2001/302—Stain compositions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The present invention relates to a kind of collagenous fibres horse pine trichrome stain kit and preparation method thereof and colouring method, staining kit includes box body, include 6 drop bottles in box body, it is respectively provided with different reagents in 6 drop bottles, the capacity of drop bottle is 5ml or 10ml, and the reagent in 6 drop bottles is respectively haematoxylin dyeing liquid, hydrochloric acid breaks up liquid, return blue liquid, Ponceaux acid fuchsin liquid, phosphomolybdic acid liquid and aniline blue liquid.The present invention is optimized from the packaging of kit, the formula of reagent and dyeing course, and then it is assembled into a set of collagenous fibres horse pine trichrome stain kit based on drip, the rapid dyeing of small-scale histotomy is can be directly used for, there is economic, practical, special efficacy, quick.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of collagenous fibres horse pine trichrome stain kit and its preparation
Method and colouring method.
Background technology
With pathological fast development, pathological tissue dyeing becomes one of the field key technology, is based particularly on group
Yihong-haematoxylin dyeing method of basic structure is knitted, not only can intuitively reflect the change of organization internal cytoplasm and nucleus, but also
Histiocytic pathological condition can be judged by the change of cell and nucleus.However, due to the complexity of cell results, tissue
Content also there are a variety of special constructions, as collagenous fibres, reticular fibre, elastic fibers, musculature, fat, glycogen, mucus,
How pathologic precipitation (such as copper, hemosiderin), nucleic acid etc., quickly define the change of special construction component in histocyte
As the field urgent problem to be solved.Therefore, histocyte specific stain technology is developed rapidly, can by the technology
Determine exotic matter, lesion and pathogen for occurring in histocyte in normal configuration and pathological change process etc., be more advantageous to
Intuitively show histiocytic Pathologic changes.
Collagenous fibres are that a kind of distribution is most extensive, content is most, and mainly contain collagen and amino acid (such as sweet ammonia
Acid, dried meat ammonia and hydroxyproline etc.) fiber composition, be distributed widely in each internal organs, it is the richest in skin, sclera and tendon
It is rich.For the pathological change of clear and definite collagenous fibres, the specific stain methods such as Van Gieson, Masson and Mallary are commonly used, it is light
Green to be dyed green, aniline blue can be dyed blueness.However, in view of diversity and the dyeing of above method staining reagent
The complexity of method so that the comparatively laborious complexity of the dyeing course, so as to limit its large-scale application clinically.At present
The commercialized complete dyeing product of in the market is almost without the reagent of Some Enterprises exploitation is mostly the part in above-mentioned colouring method
Big specification reagent, as 250ml and 500ml is packed, dyeing course relies on the equipment such as dye vat, cause dyeing process it is complicated, into
This height, so as to limit its extensive use.
The content of the invention
It is an object of the invention to provide a kind of collagenous fibres horse pine trichrome stain kit and preparation method thereof and dyeing
Method, causes the problems such as complicated, of high cost to solve to rely on dye vat dyeing in the prior art.
The present invention provides a kind of collagenous fibres horse pine trichrome stain kit, including box body, 6 drops are included in box body
Bottle, different reagents are respectively provided with 6 drop bottles, the capacity of drop bottle is 5ml or 10ml.
Further, the reagent in 6 drop bottles is respectively haematoxylin dyeing liquid, and hydrochloric acid differentiation liquid, returns blue liquid, Ponceaux acid
Property magenta liquid, phosphomolybdic acid liquid and aniline blue liquid.
Another aspect provides a kind of preparation method of collagenous fibres horse pine trichrome stain kit, including such as
Lower step：
1) preparation of reagents
A, haematoxylin dyeing liquid：Weigh quantitative hematoxylin to be dissolved in absolute ethyl alcohol, obtain solution A, weigh quantitative sulphur
Sour aluminium is dissolved in distilled water, obtains solution B, and sodium iodate is added after solution A and solution B are mixed and glacial acetic acid dissolves, most afterwards through filter
Membrane filtration；
B, hydrochloric acid differentiation liquid：Measure and mixed in quantitative hydrochloric acid addition ethanol；
C, blue liquid is returned：Weigh quantitative lithium carbonate and be dissolved in distilled water, through membrane filtration after mixing；
D, Ponceaux acid fuchsin liquid：Quantitative Ponceaux and acid fuchsin are weighed, adds in distilled water, is added after mixing
Acetic acid, most afterwards through membrane filtration；
E, phosphomolybdic acid liquid：Weigh quantitative phosphomolybdic acid and be dissolved in distilled water, through membrane filtration after mixing；
F, aniline blue liquid：Weigh quantitative aniline blue and be dissolved in distilled water, acetic acid is added, through membrane filtration after mixing；
2) reagent dispenses
Haematoxylin dyeing liquid, hydrochloric acid obtained by step 1) are broken up into liquid respectively, return blue liquid, Ponceaux acid fuchsin liquid, phosphorus molybdenum
Acid solution and aniline blue liquid are divided in drop bottle by kit specification, and the drop bottle equipped with 6 kinds of reagents finally is positioned over kit
In box body.
Further, the aperture of filter membrane is 0.45 μm.
Another aspect of the present invention provides a kind of colouring method based on collagenous fibres horse pine trichrome stain kit, including
Following steps：
1) 1-2 drop haematoxylin dyeing liquid is added dropwise on tissue sections, dyes 5-10min, washes 2-3min；
2) 1-2 drops hydrochloric acid differentiation liquid is added dropwise on step 1) products therefrom, breaks up 2-3 seconds, washes 2-3min；
3) 1-2 is added dropwise on step 2) products therefrom and returns blue liquid, return 6-10 seconds blue, washing 2-3min；
4) 1-2 drop Ponceaux acid fuchsin liquid is added dropwise on step 3) products therefrom, dyes 5-8min, washes 1-2min；
5) 1-2 drop phosphomolybdic acid liquid is added dropwise on step 4) products therefrom, dyes 1-3min, washes 2-3min；
6) 1-2 drop aniline blue liquid is added dropwise on step 5) products therefrom, is incubated 5min, washes 2-3min；
7) step 6) products therefrom is placed in 60 DEG C of incubators and dried, transparent, mounting.
Beneficial effect using the invention described above technical solution is：
The present invention is optimized from the packaging of kit, the formula of reagent and dyeing course, and then is assembled into a set of be based on
The collagenous fibres horse pine trichrome stain kit of drip, can be directly used for the rapid dyeing of small-scale histotomy, be specially：
Reagent packaging is carried out using drop bottle, has not only saved staining reagent, but also has broken traditional dye vat dye packed greatly
Color barrier, has the characteristics that economic, practical, special efficacy, quick；
System optimization exploitation is assembled into set kit, and compensate in the market Partial key staining reagent cannot complete a full set of dye
The limitation of color, the complete dyeing and displaying of collagenous fibres can be completed by the set kit.
Brief description of the drawings
Fig. 1 is collagenous fibres Ma Songsan color reagents box coloration result figure of the present invention.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical solution be clearly and completely described, it is clear that described embodiment is part of the embodiment of the present invention, rather than
Whole embodiments.
The present invention provides a kind of collagenous fibres horse pine trichrome stain kit, including box body, 6 drops are included in box body
Bottle, is respectively provided with different reagents in 6 drop bottles, and the capacity of drop bottle is 5ml or 10ml, and the reagent in 6 drop bottles is respectively bush
Plain dyeing liquor, hydrochloric acid differentiation liquid, returns blue liquid, Ponceaux acid fuchsin liquid, phosphomolybdic acid liquid and aniline blue liquid.
A kind of preparation method of collagenous fibres horse pine trichrome stain kit is present embodiments provided, is included the following steps：
1) preparation of reagents
A, haematoxylin dyeing liquid：Weigh 4g hematoxylins to be dissolved in 250ml absolute ethyl alcohols, obtain solution A, weigh 36g sulfuric acid
Aluminium is dissolved in 750ml distilled water, obtains solution B, adds 0.4g sodium iodates after solution A and solution B are mixed and appropriate glacial acetic acid is molten
Solution, most afterwards through 0.45 μm of membrane filtration；
B, hydrochloric acid differentiation liquid：Measure and mixed in the hydrochloric acid addition 99ml ethanol of 1ml；
C, blue liquid is returned：Weigh 1.3g lithium carbonates and be dissolved in 100ml distilled water, through 0.45 μm of membrane filtration after mixing；
D, Ponceaux acid fuchsin liquid：Ponceaux and the 0.3g acid fuchsins of 0.7g is weighed, is added in 99ml distilled water, is mixed
1ml acetic acid is added after even, most afterwards through 0.45 μm of membrane filtration；
E, phosphomolybdic acid liquid：Weigh 1g phosphomolybdic acids and be dissolved in 100ml distilled water, through 0.45 μm of membrane filtration after mixing；
F, aniline blue liquid：Weigh 2g aniline blues and be dissolved in 98ml distilled water, add 2ml acetic acid, through 0.45 μm of filter after mixing
Membrane filtration；
2) reagent dispenses
Haematoxylin dyeing liquid, hydrochloric acid obtained by step 1) are broken up into liquid respectively, return blue liquid, Ponceaux acid fuchsin liquid, phosphorus molybdenum
Acid solution and aniline blue liquid are divided in drop bottle by kit specification, and the drop bottle equipped with 6 kinds of reagents finally is positioned over kit
In box body.
Another embodiment of the present invention provides a kind of colouring method based on collagenous fibres horse pine trichrome stain kit, bag
Include following steps：
1) prepared by animal model
Oncomelania containing cercaria prevents research institute purchased from Jiangsu Province, China blood fluke, cercaria is escaped, through belly skin infection BALB/
C mouse, every Mouse artificial infect schistosoma japonicum cercariae 20, continue to feed 9 weeks after infection, kill mouse afterwards and take liver
Organize spare.
2) tissue sampling, fixation and embedding
In vitro tissue is put into 4% paraformaldehyde tissue fixative solution and fixes 24h-48h, fixer volume is more than organizer
4-10 times of product；By the tissue fixed into the water 30min to wash away fixer；Tissue is then put into 75% ethanol 2h, is turned
85% ethanol 2h is moved on to, goes to 95% ethanol 1h, goes to 100% ethanol 1.5-2h；Then tissue is put into transparent in dimethylbenzene I
30-45min, is transferred to transparent 30-45min in dimethylbenzene II；Tissue is put into 63 DEG C of paraffin, waxdip 1h；63 DEG C of paraffin are transferred to, are soaked
Wax 2h；The paraffin dissolved in baking oven is poured into embedded box, tissue is put into embedded box embedding.
3) histotomy
Wax stone in embedded box is put into slicer and is slightly repaiied, after tissue leaks out, slice thickness is adjusted to required size
Cut into slices, section is clamped with sharp mouth tweezer and is put into 45 DEG C or so warm water, treats that it does not have fold, is fully deployed, by slide 45
In degree insertion water, move under optimal section and gently pick up, section is transferred on slide；Section is fitted into slide holding frame, 65
0.5-1h is placed in DEG C baking oven；Section is put into dimethylbenzene I 5-10min that dewaxes, is transferred in dimethylbenzene II 5-10min that dewaxes；
Absolute ethyl alcohol washs 1-5min, 95% ethanol washing 1-5min, 75% ethanol washing 1-5min, finally washing section.
4) dye
A, 1 drop haematoxylin dyeing liquid is added dropwise on tissue sections, dyes 5-10min, washes 2-3min；
B, 1 drop hydrochloric acid differentiation liquid is added dropwise on step a) products therefroms, breaks up 2-3 seconds, washes 2-3min；
C, 1 is added dropwise on step b) products therefroms and returns blue liquid, return 6-10 seconds blue, washing 2-3min；
D, 1 drop Ponceaux acid fuchsin liquid is added dropwise on step c) products therefroms, dyes 5-8min, washes 1-2min；
E, 1 drop phosphomolybdic acid liquid is added dropwise on step d) products therefroms, dyes 1-3min, washes 2-3min；
F, 1 drop aniline blue liquid is added dropwise on step e) products therefroms, is incubated 5min, washes 2-3min；
G, step f) products therefroms are placed in 60 DEG C of incubators and dried, transparent, mounting.
Fig. 1 is the embodiment coloration result figure, it is seen that worm's ovum area hepatic fibrosis-renal tubular ectasia syndrome is obvious, and coloring is excellent.
To sum up, the present invention is optimized from the packaging of kit, the formula of reagent and dyeing course, and then is assembled into a set of
Collagenous fibres horse pine trichrome stain kit based on drip, can be directly used for the rapid dyeing of small-scale histotomy；
Reagent packaging is carried out using drop bottle, has not only saved staining reagent, but also has broken traditional dye vat dye packed greatly
Color barrier, has the characteristics that economic, practical, special efficacy, quick；
System optimization exploitation is assembled into set kit, and compensate in the market Partial key staining reagent cannot complete a full set of dye
The limitation of color, the complete dyeing and displaying of collagenous fibres can be completed by the set kit.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic into
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Claims (5)
1. a kind of collagenous fibres horse pine trichrome stain kit, it is characterised in that include 6 drops including box body, in the box body
Bottle, different reagents are respectively provided with 6 drop bottles, the capacity of the drop bottle is 5ml or 10ml.
2. collagenous fibres horse pine trichrome stain kit according to claim 1, it is characterised in that in 6 drop bottles
Reagent be respectively haematoxylin dyeing liquid, hydrochloric acid differentiation liquid, returns blue liquid, Ponceaux acid fuchsin liquid, phosphomolybdic acid liquid and aniline blue
Liquid.
A kind of 3. preparation method of the collagenous fibres horse pine trichrome stain kit of claim 1 or 2, it is characterised in that bag
Include following steps：
1) preparation of reagents
A, haematoxylin dyeing liquid：Weigh quantitative hematoxylin to be dissolved in absolute ethyl alcohol, obtain solution A, weigh quantitative aluminum sulfate
It is dissolved in distilled water, obtains solution B, sodium iodate is added after solution A and solution B are mixed and glacial acetic acid dissolves, most afterwards through filter membrane mistake
Filter；
B, hydrochloric acid differentiation liquid：Measure and mixed in quantitative hydrochloric acid addition ethanol；
C, blue liquid is returned：Weigh quantitative lithium carbonate and be dissolved in distilled water, through membrane filtration after mixing；
D, Ponceaux acid fuchsin liquid：Quantitative Ponceaux and acid fuchsin are weighed, adds in distilled water, second is added after mixing
Acid, most afterwards through membrane filtration；
E, phosphomolybdic acid liquid：Weigh quantitative phosphomolybdic acid and be dissolved in distilled water, through membrane filtration after mixing；
F, aniline blue liquid：Weigh quantitative aniline blue and be dissolved in distilled water, acetic acid is added, through membrane filtration after mixing；
2) reagent dispenses
Haematoxylin dyeing liquid, hydrochloric acid obtained by the step 1) are broken up into liquid respectively, return blue liquid, Ponceaux acid fuchsin liquid, phosphorus molybdenum
Acid solution and aniline blue liquid are divided in drop bottle by kit specification, and the drop bottle equipped with 6 kinds of reagents finally is positioned over kit
In box body.
4. the preparation method of collagenous fibres horse pine trichrome stain kit according to claim 3, it is characterised in that described
The aperture of filter membrane is 0.45 μm.
5. a kind of colouring method based on the collagenous fibres horse pine trichrome stain kit of claim 1 or 2, its feature exist
In including the following steps：
1) 1-2 drop haematoxylin dyeing liquid is added dropwise on tissue sections, dyes 5-10min, washes 2-3min；
2) 1-2 drops hydrochloric acid differentiation liquid is added dropwise on step 1) products therefrom, breaks up 2-3 seconds, washes 2-3min；
3) 1-2 is added dropwise on step 2) products therefrom and returns blue liquid, return 6-10 seconds blue, washing 2-3min；
4) 1-2 drop Ponceaux acid fuchsin liquid is added dropwise on step 3) products therefrom, dyes 5-8min, washes 1-2min；
5) 1-2 drop phosphomolybdic acid liquid is added dropwise on step 4) products therefrom, dyes 1-3min, washes 2-3min；
6) 1-2 drop aniline blue liquid is added dropwise on step 5) products therefrom, is incubated 5min, washes 2-3min；
7) step 6) products therefrom is placed in 60 DEG C of incubators and dried, transparent, mounting.
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