CN116558928A - Red dye liquor for Masson trichromatic dyeing, reagent combination and method - Google Patents
Red dye liquor for Masson trichromatic dyeing, reagent combination and method Download PDFInfo
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- 238000004043 dyeing Methods 0.000 title claims abstract description 64
- 239000001044 red dye Substances 0.000 title claims abstract description 34
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 17
- 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 claims abstract description 86
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 67
- 239000000975 dye Substances 0.000 claims abstract description 38
- 239000001045 blue dye Substances 0.000 claims abstract description 37
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 102000008186 Collagen Human genes 0.000 claims abstract description 19
- 108010035532 Collagen Proteins 0.000 claims abstract description 19
- 229920001436 collagen Polymers 0.000 claims abstract description 19
- 239000002904 solvent Substances 0.000 claims abstract description 15
- 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 13
- 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 12
- HSXUHWZMNJHFRV-UHFFFAOYSA-L disodium;6-oxido-5-phenyldiazenyl-4-sulfonaphthalene-2-sulfonate Chemical class [Na+].[Na+].OC1=CC=C2C=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1N=NC1=CC=CC=C1 HSXUHWZMNJHFRV-UHFFFAOYSA-L 0.000 claims abstract description 10
- MBXSHBIQMDKTEW-UHFFFAOYSA-N 3-bromobutanenitrile Chemical compound CC(Br)CC#N MBXSHBIQMDKTEW-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000007787 solid Substances 0.000 claims abstract description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 56
- 239000000243 solution Substances 0.000 claims description 45
- 210000001519 tissue Anatomy 0.000 claims description 38
- 238000010186 staining Methods 0.000 claims description 34
- 229910052742 iron Inorganic materials 0.000 claims description 28
- 238000005406 washing Methods 0.000 claims description 16
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 210000001072 colon Anatomy 0.000 claims description 7
- 210000005228 liver tissue Anatomy 0.000 claims description 6
- 210000005084 renal tissue Anatomy 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 4
- 239000012192 staining solution Substances 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000000887 hydrating effect Effects 0.000 claims description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 210000003205 muscle Anatomy 0.000 claims 1
- 210000001087 myotubule Anatomy 0.000 abstract description 20
- 239000000835 fiber Substances 0.000 abstract description 19
- 230000004069 differentiation Effects 0.000 abstract description 8
- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 abstract description 3
- 229960001500 bivalirudin Drugs 0.000 abstract description 3
- 108010055460 bivalirudin Proteins 0.000 abstract description 3
- 229960000583 acetic acid Drugs 0.000 description 17
- 230000000694 effects Effects 0.000 description 17
- 239000000203 mixture Substances 0.000 description 11
- 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 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000009472 formulation Methods 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 239000008213 purified water Substances 0.000 description 5
- 239000008399 tap water Substances 0.000 description 5
- 235000020679 tap water Nutrition 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- 240000007263 Pinus koraiensis Species 0.000 description 2
- 235000011615 Pinus koraiensis Nutrition 0.000 description 2
- YJVBLROMQZEFPA-UHFFFAOYSA-L acid red 26 Chemical compound [Na+].[Na+].CC1=CC(C)=CC=C1N=NC1=C(O)C(S([O-])(=O)=O)=CC2=CC(S([O-])(=O)=O)=CC=C12 YJVBLROMQZEFPA-UHFFFAOYSA-L 0.000 description 2
- 239000000987 azo dye Substances 0.000 description 2
- WZRZTHMJPHPAMU-UHFFFAOYSA-L disodium;(3e)-3-[(4-amino-3-sulfonatophenyl)-(4-amino-3-sulfophenyl)methylidene]-6-imino-5-methylcyclohexa-1,4-diene-1-sulfonate Chemical compound [Na+].[Na+].C1=C(S([O-])(=O)=O)C(=N)C(C)=CC1=C(C=1C=C(C(N)=CC=1)S([O-])(=O)=O)C1=CC=C(N)C(S(O)(=O)=O)=C1 WZRZTHMJPHPAMU-UHFFFAOYSA-L 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000010827 pathological analysis Methods 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 239000004214 Fast Green FCF Substances 0.000 description 1
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019240 fast green FCF Nutrition 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
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- 230000035515 penetration Effects 0.000 description 1
- 230000036573 scar formation Effects 0.000 description 1
- 230000001360 synchronised effect Effects 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- 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)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a red dye solution, a reagent combination and a method for Masson trichromatic dyeing, and belongs to the technical field of histochemistry. The red dye liquor comprises 0.4 to 1.0 percent of bivalirudin scarlet, 0.4 to 1.0 percent of allure red, 1 to 2 percent of acetic acid, water as solvent and further comprises 0.05 to 0.15 percent of orange G sodium salt; the reagent combination comprises the red dye liquor and also comprises a blue dye liquor, wherein the blue dye liquor comprises 2% of aniline blue, 1% -2% of acetic acid, the solvent is water, and further comprises 0.005% -0.03% of solid green, 0.0025% -0.015% of bright green or 0.005% -0.03% of bromocresol green sodium salt. The reagent combination also comprises hematoxylin dye solution, red differentiating agent and blue differentiating agent. The improved red dye liquor for Masson trichromatic dyeing provided by the invention increases the affinity for dyeing the muscle fiber, so that the dye liquor is more firm in dyeing with the muscle fiber, and can still keep bright red after phosphomolybdic acid differentiation. Meanwhile, the improved blue dye liquor dyes the collagen fiber more brightly, so that the color tone contrast between the muscle fiber and the collagen fiber is more obvious and easier to distinguish.
Description
Technical Field
The invention relates to the field of histochemistry, in particular to a red dye solution, a reagent combination and a method for Masson trichromatic dyeing.
Background
The Masson staining method is commonly used for identifying and staining collagen fibers and muscle fibers, and is one of important methods for pathological tissue preparation. Collagen fibers can appear in the body along with the development of pathological processes in chronic inflammation, organization and scar formation. These fibers are often difficult to identify from fibrin in early HE-stained sections, but can be confirmed by Masson staining.
At present, the Masson dyeing method mainly uses combination of ponceau and acid fuchsin to dye myofibers, and further can add bikini scarlet; collagen fibers were stained with aniline blue. For example, chinese patent No. CN110926911a discloses a dyeing kit of pinus koraiensis and a dyeing method thereof, the dyeing kit of pinus koraiensis comprises ponceau acid fuchsin dye liquor and aniline blue dye liquor, and the preparation method of ponceau acid fuchsin dye liquor comprises: dissolving 4g of ponceau and 2g of acid fuchsin in 495mL of distilled water, and adding 5mL of glacial acetic acid to obtain ponceau acid fuchsin dye liquor; the preparation method of the aniline blue dye liquor comprises the following steps: 10g of aniline blue is dissolved in 490mL of distilled water to obtain solution B, and 10mL of glacial acetic acid is added to the solution B to obtain aniline blue dye solution. As another example, weiyong et al disclose an improved Masson trichromatic dyeing process, wherein the dye liquor is: 30mL of l% ponceau 2R aqueous solution, 20mL of 1% acid fuchsin aqueous solution, 50mL of l% BILIMISSID aqueous solution, and 1mL of glacial acetic acid (Weiyong, yang Qun, fan Xiaoli; an improved Masson trichromatic staining method [ J ]; journal of diagnostic pathology; 2005).
However, since ponceau and acid fuchsin are not firm enough to dye the muscle fibers, and are easy to decolorize by differentiation or washing, the conventional Masson trichromatic dyeing causes dark and light red to dye the muscle fibers, cytoplasm and the like, and the color contrast of the tissue structure is not bright enough or is not easy to distinguish, and the red is easy to be covered after counterstaining blue, becomes darker, bluish purple and even cannot be distinguished.
Disclosure of Invention
In order to solve at least one of the technical problems, the invention aims to provide a Masson trichromatic dye solution combination which is bright and firm in dyeing, clear in tissue structure color contrast and easy to distinguish, and the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a red dye solution for Masson trichromatic dyeing, which comprises 0.4-1.0% of Biglichid by mass and volume fraction, 0.4-1.0% of alluring red by mass and volume fraction and 1-2% of acetic acid by volume fraction, wherein the solvent is water.
In the present invention, instead of ponceau and acid fuchsin, there are used specific brinzee and allure which are azo dyes having a molecular weight of 400 to 600, which have a more firm dyeing effect on muscle fibers than ponceau and acid fuchsin, and which make the tissue-colored red more vivid and more vivid contrast with blue.
In some embodiments of the invention, the red dye liquor further comprises 0.05-0.15% of orange G sodium salt by mass and volume fraction. The orange G sodium salt is orange azo dye with the molecular weight of 400-600, can adjust the condition that the red dye is used for dyeing the muscle fiber too deeply, and can prevent the superposition of red dye liquor and blue dye liquor on tissues from being dyed too deeply or too darkly by utilizing the orange G sodium salt to carry out color mixing on the red, so that the red dyeing of the muscle fiber is more obviously compared with the blue dyeing of the collagen fiber.
The second aspect of the invention provides a reagent combination for Masson trichromatic dyeing, which comprises the red dye liquor according to any one of the first aspect of the invention, and further comprises a blue dye liquor, wherein the blue dye liquor comprises 2% of aniline blue by mass and volume fraction and 1% -2% of acetic acid by volume fraction, and the solvent is water.
In some embodiments of the invention, the blue dye liquor further comprises solid green with a mass-volume fraction of 0.005% to 0.03%, bright green with a mass-volume fraction of 0.0025% to 0.015%, or bromocresol green sodium salt with a mass-volume fraction of 0.005% to 0.03%. The blue dye liquor of aniline is tinted by utilizing solid green or bright green or bromocresol green sodium salt, so that the blue dye liquor can be prevented from dyeing collagen fibers too deeply.
Further, the reagent combination further comprises hematoxylin dye solution, red differentiating agent and/or blue differentiating agent.
In some embodiments of the invention, the hematoxylin dye liquor is Weibert iron hematoxylin dye liquor, the Weibert iron hematoxylin dye liquor is formed by mixing Weibert iron hematoxylin A liquor and Weibert iron hematoxylin B liquor according to the proportion of 1:2-2:1,
the Weibert iron hematoxylin A solution comprises 0.5-2.0% of hematoxylin by mass and volume fraction, 10-20% of glycol by volume fraction and 95% of ethanol by solvent;
the Weibert iron hematoxylin B solution comprises 0.36 to 0.38 percent of HCl and 1.0 to 1.5 percent of FeCl by mass percent 3 The solvent is water.
In some embodiments of the present invention,
the preparation method of 50mL Weibert iron hematoxylin A solution comprises the following steps: 0.5g of hematoxylin is weighed, 7mL of ethylene glycol is added, and 43mL of 95% ethanol is added and mixed uniformly.
The preparation method of 50mL Weibert iron hematoxylin B solution comprises the following steps: concentrated hydrochloric acid 0.5mL was dissolved in 50mL of purified water, and 0.58g of anhydrous ferric trichloride was added and mixed well. Wherein the mass fraction of the concentrated hydrochloric acid is 36-38%.
In some preferred embodiments of the invention, the Weiert iron hematoxylin dye solution is formed by mixing Weiert iron hematoxylin A solution and Weiert iron hematoxylin B solution according to a ratio of 1:1.
In some embodiments of the invention, the red differentiating agent is a phosphotungstic phosphomolybdic acid solution, the phosphotungstic phosphomolybdic acid solution comprises 2.0-3.0% of phosphotungstic acid by mass and 2.0-3.0% of phosphomolybdic acid by mass and volume, and the solvent is water.
In some embodiments of the invention, 100mL of the phosphotungstic phosphomolybdic acid solution is formulated by: 2.5g of phosphotungstic acid and 2.5g of phosphomolybdic acid are weighed, and 100mL of water is added for dissolution.
In some embodiments of the invention, the blue differentiating agent is an acetic acid solution comprising 0.5% -2% acetic acid by volume and the solvent is water.
In some embodiments of the invention, 500mL of the acetic acid solution is formulated by: 5mL of acetic acid was added to 500mL of purified water and mixed well.
In a third aspect, the invention provides the use of a combination of reagents for Masson trichromatic staining according to any of the second aspects of the invention in the preparation of a kit for trichromatic staining of tissue.
The trichromatic staining refers to the staining of nuclei and the selective display of collagen fibers and muscle fibers. Trichromatic staining is related to the size of the anionic dye molecules and tissue penetration: the size of the molecules is represented by the molecular weight, small molecular weight is easy to penetrate through tissue with compact structure and low permeability, and large molecular weight can only enter into tissue with loose structure and high permeability. However, aniline blue has a large molecular weight, so that after trichromatic dyeing, myofibers and cytoplasm are red, collagen fibers are blue, and cell nuclei are purplish brown or blue brown, and trichromatic dyeing is mainly used for distinguishing collagen fibers from myofibers, so that a tissue mechanism is easy to distinguish.
In the present invention, the tissue is a tissue including collagen fibers and/or muscle fibers. The modified Masson trichromatic staining solution is used for staining collagen fibers and muscle fibers, and can be used for judging and analyzing the fibrous tissue morphology, fibrotic lesions and the like of organs such as heart, kidney, liver and the like.
In some embodiments of the invention, the tissue is selected from at least one of the group consisting of cervical tissue, colon tissue, kidney tissue, liver tissue.
A third aspect of the present invention provides a method for trichromatic staining of tissue with a reagent combination comprising a red dye solution, a blue dye solution, a hematoxylin dye solution, a red differentiating agent, and a blue differentiating agent according to the second aspect of the present invention, comprising the steps of:
s1, heating and roasting slices carrying tissue samples, and dewaxing and hydrating;
s2, staining the tissue for 5-10 minutes by using hematoxylin dye liquor, and washing off redundant hematoxylin dye liquor;
s3, using the red dye liquor to incubate and dye for 5-10 minutes, and washing off redundant red dye liquor;
s4, differentiating for 1-3 minutes by using the red differentiating agent, and washing off superfluous red differentiating agent;
s5, using the blue dye liquor to incubate and dye for 5-10 minutes, and washing off the redundant blue dye liquor;
s6, differentiating for 0.5-2 minutes by using the blue differentiating agent, and washing away the superfluous blue differentiating agent;
s7, drying slices after dehydrating and transparentizing, sealing the slices with neutral resin, and observing the dyeing condition under a microscope.
The hematoxylin dye liquor is Weibert iron hematoxylin dye liquor, and the Weibert iron hematoxylin dye liquor is formed by mixing Weibert iron hematoxylin A liquor and Weibert iron hematoxylin B liquor in a ratio of 1:2-2:1 before use.
The beneficial effects of the invention are that
Compared with the prior art, the invention has the following beneficial effects:
the red dye liquor for Masson trichromatic dyeing of the invention uses brinzee scarlet, allure red instead of ponceau and acid fuchsine, so that the red and blue of tissue coloring are more vivid in contrast. Further, the orange G sodium salt is used for toning the red, so that the superposition dyeing of the red dye liquor and the blue dye liquor on tissues is effectively prevented from being too deep or too dark. Furthermore, the blue dye liquor in the reagent combination for Masson trichromatic dyeing uses solid green or bright green or bromocresol green sodium salt to carry out color matching on the aniline blue dye liquor, so that the blue dye liquor is effectively prevented from dyeing the collagen fibers too deeply.
The improved red dye liquor for Masson trichromatic dyeing provided by the invention increases the affinity for dyeing the muscle fiber, so that the dye liquor is more firm in dyeing with the muscle fiber, and can still keep bright red after phosphomolybdic acid differentiation. Meanwhile, the improved blue dye liquor dyes the collagen fiber more brightly, so that the color tone contrast between the muscle fiber and the collagen fiber is more obvious and easier to distinguish.
Drawings
FIG. 1 is a graph showing different staining effects on liver tissue in an embodiment of the present invention. Wherein A: a staining result labeled "0"; b: a staining result marked "-1"; c: staining results labeled "+1"; d: staining results labeled "+2"; e: staining results marked "+3".
FIG. 2 is a graph showing the different staining effects on kidney tissue in an embodiment of the present invention. Wherein A: a staining result labeled "0"; b: a staining result marked "-1"; c: staining results labeled "+1"; d: staining results labeled "+2"; e: staining results marked "+3". .
Detailed Description
Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited are incorporated by reference, particularly as they relate to the definitions of terms in the art. If the definition of a particular term disclosed in the prior art does not conform to any definition provided in this application, the definition of that term provided in this application controls.
Numerical ranges in this application are approximations, so that it may include the numerical values outside of the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value in 1 unit increase, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.
The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or procedure, and are not related to whether or not such other component, step or procedure is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising," "including," or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant, or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.
In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.
Examples
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1 formulation optimization of Masson Red dye liquor
1. Red and blue dye liquor preparation
This example provides 16 Masson red dye formulas A0-a15, and 2 control red dye formulas as shown in table 1:
table 1 100mL red dye liquor formulation
Wherein, RA1 adopts the formula disclosed in Chinese invention patent CN 110926911A; RA2 uses the formula described in reference (Weiyong, yang Qun, fan Xiaoli; introduction of a modified Masson trichromatography [ J ]; J. Diagnostic pathology; 2005 01); a0 is a formulation using bivalirudin scarlet instead of ponceau in RA 2.
The blue dye solutions are all prepared from the formula disclosed in Chinese patent No. CN110926911A, and the number is RB1.
RB1: weigh 2g aniline blue and 2mL acetic acid, add water to 100mL.
Other reagents and corresponding preparation methods are as follows:
weibert iron hematoxylin A solution: 0.5g of hematoxylin is weighed, 7mL of ethylene glycol is added, and 43mL of 95% ethanol is added and mixed uniformly for standby.
Weibert iron hematoxylin B solution: concentrated hydrochloric acid 0.5mL is dissolved in 50mL of purified water, and 0.58g of anhydrous ferric trichloride is added and mixed uniformly for standby.
The Weibert iron hematoxylin A solution and the Weibert iron hematoxylin B solution are mixed in equal proportion before use, and the Weibert iron hematoxylin dye solution is obtained.
Phosphotungstic phosphomolybdic acid solution (red differentiating agent): 2.5g of phosphotungstic acid and 2.5g of phosphomolybdic acid are weighed, 100mL of purified water is added, and the mixture is dissolved for standby.
Acetic acid solution (blue differentiating agent): 5mL of acetic acid is added into 500mL of purified water and mixed well for standby.
2. Test organization
Each red dye (RA 1, RA2, R0-R15) was tested in combination with RB1 composition dye, each dye combination testing 4 tissues: cervical, colon, kidney, liver; the loading of each slide was 0.2mL (preferably, the tissue was completely covered).
3. Test method
(1) The slice carrying the tissue sample is heated to bake the slice, and dewaxed and hydrated.
(2) The mixture was stained with Weiert iron hematoxylin dye for 8 minutes, and excess Weiert iron hematoxylin dye was removed by washing with tap water.
(3) The red dye solution is used for incubation and dyeing for 8 minutes, and the excess red dye solution is removed by washing with tap water.
(4) The red differentiation agent (phosphotungstic phosphomolybdic acid solution) was used for differentiation for 2 minutes, and tap water was used for washing to remove the excess red differentiation agent.
(5) The blue dye solution is used for incubation and dyeing for 8 minutes, and the excess blue dye solution is removed by tap water washing.
(6) The mixture was differentiated for 1 minute using a blue differentiation agent (acetic acid solution), and the excess blue differentiation agent was removed by washing with tap water.
Finally, the slices are dried and dried after being dehydrated and transparent, and then the slices are sealed by neutral resin, and the dyeing condition is observed under a microscope.
4. Test results
The test results are shown in Table 2:
TABLE 2 results of dyeing with each Red dye liquor and RB1 combination
| Red dye liquor numbering | Cervical of the uterus | Colon | Kidney and kidney | Liver | Total score |
| RA1 | 0 | 0 | 0 | 0 | 0 |
| RA2 | +1 | 0 | +1 | 0 | +2 |
| A0 | 0 | 0 | -1 | -1 | -2 |
| A1 | 0 | 0 | 0 | 0 | 0 |
| A2 | +1 | +1 | 0 | +1 | +3 |
| A3 | +1 | +1 | +1 | +1 | +4 |
| A4 | +1 | +1 | +1 | +1 | +4 |
| A5 | +1 | 0 | +1 | +1 | +3 |
| A6 | +1 | +1 | +1 | +1 | +4 |
| A7 | +1 | +1 | +1 | +1 | +4 |
| A8 | 0 | 0 | 0 | +1 | +1 |
| A9 | +1 | +1 | +1 | +1 | +4 |
| A10 | +2 | +2 | +2 | +2 | +8 |
| A11 | +2 | +2 | +2 | +2 | +8 |
| A12 | +2 | +2 | +1 | +2 | +7 |
| A13 | +2 | +2 | +2 | +2 | +8 |
| A14 | +1 | +1 | +1 | +1 | +4 |
| A15 | +2 | +2 | +2 | +2 | +8 |
And (3) result judgment:
collagen fibers are blue, muscle fibers and cytoplasm are red, nuclei are purplish brown or blue brown, and red blood cells are orange red. Based on the conventional formulation (RA 1+ RB 1), labeled 0, with the optimization label +, the degradation label-, the magnitude of the optimization/degradation degree is represented by a number, namely +1, +2 … …, -1, -2 … …. The optimization means that the dyeing color is bright, the contrast is more vivid, the tissue and cell structure dyeing can be obviously distinguished, and the discrimination is easier; degradation is too shallow and too dark, and the contrast is reduced, so that the tissue and cell structure are not sufficiently distinguished by dyeing, and are not easy to distinguish. For liver tissue, the staining results for markers 0, +1, +2, +3, -1 are shown in FIG. 1; for kidney tissue, the staining results for markers 0, +1, +2, +3, -1 are shown in FIG. 2.
As can be seen from Table 2, RA2 did not improve the staining effect in colon and liver tissue relative to RA1 (0), and the staining effect in cervical and kidney tissue was optimized (+1), indicating that addition of BIBrazil scarlet to ponceau and acid fuchsine improved the staining effect, but the improvement was not very pronounced. If ponceau is further replaced with bivalirudin (A0), the staining effect is not improved in cervical and colon tissues (0), and the staining effect is even deteriorated in kidney and liver tissues (-1).
If ponceau red and acid fuchsin are replaced by bivalirus scarlet and allure red, the dyeing results of different tissues are optimized to different degrees under different adding proportions, the dyeing of the myofibers is bright, the dyeing contrast of the collagen fibers and the collagen fibers is obvious and easy to distinguish, and the tissue and cell structures are clear and distinguishable, so that the dyeing effect is obviously better than that before the optimization. However, if the ratio of the confusing red to the confusing red is more than 2.5 times, or the ratio of the confusing red to the confusing red is more than 2.5 times, the dyeing effect is hardly optimized (e.g., A1 and A8).
Based on the comparison of the brinzhong and the allure red, the inventor further adds orange G sodium salt for testing, and discovers that after the orange G sodium salt is added, the dyeing effect is further optimized, when the addition amount of the orange G sodium salt is 0.05-0.15G, the dyeing effect is obviously improved, for example, when A10-A13 (the dyeing result is marked as +2), A9 and A14 are respectively combined with RB1, the dyeing effect improvement is not ideal (the dyeing result is marked as +1), and the dyeing effect is influenced by the excessive or the insufficient addition amount of the orange G sodium salt. As is clear from the dyeing result of A15, the reduction of the amount of acetic acid has no effect on the dyeing result, indicating that the preferred red dye liquor of the present embodiment can reduce the amount of acetic acid used.
Example 2 preparation of blue dye liquor
To further enhance the dyeing effect, the inventors optimized blue dyeing, and for this purpose, provided 15 Masson blue dye formulas B1-B15, using RB1 as a control as described above. The formulation of each blue dye liquor is shown in Table 3:
TABLE 3 100mL blue dye liquor formulation
Each blue dye liquor was tested separately in combination with the a11 composition dye liquor of example 1, test organization and method as in example 1. The staining results are shown in table 4:
TABLE 4 dyeing results for each blue dye liquor and A11 combination
| Blue dye liquor numbering | Cervical of the uterus | Colon | Kidney and kidney | Liver | Total score |
| RB1 | +2 | +2 | +2 | +2 | +8 |
| B1 | +3 | +2 | +2 | +3 | +10 |
| B2 | +3 | +3 | +3 | +3 | +12 |
| B3 | +3 | +3 | +3 | +3 | +12 |
| B4 | +3 | +3 | +2 | +3 | +11 |
| B5 | +3 | +2 | +3 | +2 | +10 |
| B6 | +3 | +2 | +3 | +2 | +10 |
| B7 | +3 | +3 | +3 | +3 | +12 |
| B8 | +3 | +3 | +2 | +3 | +11 |
| B9 | +3 | +2 | +2 | +3 | +10 |
| B10 | +3 | +3 | +3 | +3 | +12 |
| B11 | +3 | +3 | +2 | +3 | +11 |
| B12 | +3 | +2 | +2 | +3 | +10 |
| B13 | +3 | +3 | +3 | +3 | +12 |
| B14 | +3 | +3 | +3 | +3 | +12 |
| B15 | +3 | +3 | +3 | +3 | +12 |
As can be seen from Table 4, the addition of appropriate amounts of fast green FCF (B1-B4), bright green (B5-B8) or bromocresol green sodium salt (B9-B12) to aniline blue can further improve the dyeing effect to different extents, and the dyeing optimization of different tissues even reaches +3 (as can be seen from the dyeing results of B13-B15), the reduction of the acetic acid consumption has no influence on the dyeing results, which indicates that the preferred blue dye liquor can reduce the consumption of acetic acid.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. The red dye solution for Masson trichromatic dyeing is characterized by comprising 0.4-1.0% of specific brinzhong by mass and volume fraction, 0.4-1.0% of allure red by mass and volume fraction and 1-2% of acetic acid by volume fraction, wherein the solvent is water.
2. The red dye liquor for Masson trichromatic dyeing according to claim 1, further comprising 0.05-0.15% of orange G sodium salt by mass-volume fraction.
3. A reagent combination for Masson trichromatic dyeing, which is characterized by comprising the red dye liquor according to claim 1 or 2 and further comprising a blue dye liquor, wherein the blue dye liquor comprises 2% of aniline blue by mass and 2% of acetic acid by volume, and the solvent is water.
4. A reagent combination for Masson trichromatic dyeing according to claim 3, characterized in that the blue dye liquor further comprises solid green with a mass-volume fraction of 0.005% to 0.03%, bright green with a mass-volume fraction of 0.0025% to 0.015% or bromocresol green sodium salt with a mass-volume fraction of 0.005% to 0.03%.
5. The combination of reagents for Masson trichromatic staining according to claim 4, further comprising hematoxylin dye, red differentiating agent and/or blue differentiating agent.
6. The reagent combination for Masson trichromatic staining according to claim 5, wherein the hematoxylin staining solution is Weibert iron hematoxylin staining solution, the Weibert iron hematoxylin staining solution is formed by mixing Weibert iron hematoxylin A solution and Weibert iron hematoxylin B solution according to a ratio of 1:2-2:1,
the Weibert iron hematoxylin A solution comprises 0.5-2.0% of hematoxylin by mass and volume fraction, 10-20% of glycol by volume fraction and 95% of ethanol by solvent;
the Weibert iron hematoxylin B solution comprises a matrixHCl with the weight fraction of 0.36-0.38% and FeCl with the mass volume fraction of 1.0-1.5% 3 The solvent is water, and the solvent is water,
the red differentiating agent is phosphotungstic phosphomolybdic acid solution, the phosphotungstic phosphomolybdic acid solution comprises 2.0-3.0 percent of phosphotungstic acid with mass volume fraction and 2.0-3.0 percent of phosphomolybdic acid with mass volume fraction, the solvent is water,
the blue differentiating agent is acetic acid solution, the acetic acid solution comprises acetic acid with the volume fraction of 0.5% -2%, and the solvent is water.
7. Use of a combination of reagents for Masson trichromatic staining according to any of claims 3-6 for the preparation of a kit for trichromatic staining of tissues.
8. The use according to claim 7, wherein the tissue is a tissue comprising collagen fibres and/or muscle fibres.
9. The use according to claim 8, wherein the tissue is selected from at least one of the group comprising cervical tissue, colon tissue, kidney tissue, liver tissue.
10. A method of trichromatizing tissue using a Masson trichromatic stain combination of claim 6, comprising the steps of:
s1, heating and roasting slices carrying tissue samples, and dewaxing and hydrating;
s2, dyeing the tissue for 5-10 minutes by using the hematoxylin dye liquor, and washing off the redundant hematoxylin dye liquor;
s3, using the red dye liquor to incubate and dye for 5-10 minutes, and washing off redundant red dye liquor;
s4, differentiating for 1-3 minutes by using the red differentiating agent, and washing off superfluous red differentiating agent;
s5, using the blue dye liquor to incubate and dye for 5-10 minutes, and washing off the redundant blue dye liquor;
s6, differentiating for 0.5-2 minutes by using the blue differentiating agent, and washing away the superfluous blue differentiating agent;
s7, drying slices after dehydrating and transparentizing, sealing the slices with neutral resin, and observing the dyeing condition under a microscope.
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| CN117363051B (en) * | 2023-12-08 | 2024-03-05 | 北京索莱宝科技有限公司 | Collagen fiber coloring agent and application thereof |
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