CN112539986B - Ferrous sulfate reagent and kit for melanin dyeing - Google Patents
Ferrous sulfate reagent and kit for melanin dyeing Download PDFInfo
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- CN112539986B CN112539986B CN202011398959.7A CN202011398959A CN112539986B CN 112539986 B CN112539986 B CN 112539986B CN 202011398959 A CN202011398959 A CN 202011398959A CN 112539986 B CN112539986 B CN 112539986B
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- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 title claims abstract description 99
- 235000003891 ferrous sulphate Nutrition 0.000 title claims abstract description 90
- 239000011790 ferrous sulphate Substances 0.000 title claims abstract description 90
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 title claims abstract description 90
- 229910000359 iron(II) sulfate Inorganic materials 0.000 title claims abstract description 90
- 238000004043 dyeing Methods 0.000 title claims abstract description 75
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 55
- 108010010803 Gelatin Proteins 0.000 claims abstract description 24
- 239000008273 gelatin Substances 0.000 claims abstract description 24
- 229920000159 gelatin Polymers 0.000 claims abstract description 24
- 235000019322 gelatine Nutrition 0.000 claims abstract description 24
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 24
- 239000003223 protective agent Substances 0.000 claims abstract description 20
- 239000012153 distilled water Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims abstract description 17
- 239000002253 acid Substances 0.000 claims abstract description 14
- 230000001575 pathological effect Effects 0.000 claims abstract description 11
- 230000001590 oxidative effect Effects 0.000 claims abstract description 8
- 239000000049 pigment Substances 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 7
- 208000030381 cutaneous melanoma Diseases 0.000 claims abstract description 4
- 230000003902 lesion Effects 0.000 claims abstract description 4
- 201000003708 skin melanoma Diseases 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 113
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 52
- 229960000583 acetic acid Drugs 0.000 claims description 26
- 239000012362 glacial acetic acid Substances 0.000 claims description 26
- -1 potassium ferricyanide acetic acid Chemical compound 0.000 claims description 26
- 238000010186 staining Methods 0.000 claims description 26
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- 230000000052 comparative effect Effects 0.000 claims description 14
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 239000012192 staining solution Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 230000000684 melanotic effect Effects 0.000 claims description 4
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical class OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 claims description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 3
- 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 description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
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- 239000011260 aqueous acid Substances 0.000 claims 1
- 230000007170 pathology Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 201000001441 melanoma Diseases 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 description 13
- 239000000975 dye Substances 0.000 description 10
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 8
- 229910001448 ferrous ion Inorganic materials 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 239000000835 fiber Substances 0.000 description 5
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- 206010063659 Aversion Diseases 0.000 description 1
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- 230000002378 acidificating effect Effects 0.000 description 1
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- 125000000129 anionic group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 230000002197 limbic effect Effects 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- LDQICAMJIICDLF-UHFFFAOYSA-N potassium;iron(2+);iron(3+);hexacyanide Chemical compound [K+].[Fe+2].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] LDQICAMJIICDLF-UHFFFAOYSA-N 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 238000011282 treatment Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 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
- G01N1/31—Apparatus therefor
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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Abstract
The invention relates to a ferrous sulfate reagent and a kit for melanin dyeing, and the preparation method of the ferrous sulfate reagent provided by the invention comprises the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adding non-oxidizing acid to adjust the pH value to 2.0-2.5, adding a dyeing protective agent, fully stirring to dissolve the ferrous sulfate, and fixing the volume of the solution to 100 mL; the dyeing protective agent is gelatin with the mass fraction of 0.1-1%. A small amount of non-oxidative acid and a dyeing protective agent are added into the reagent to prolong the preservation time of the reagent and ensure the dyeing effect, the preparation is simple, the components of the protective agent are simple and easy to obtain, the protection effect on the ferrous sulfate dyeing effect is good, and the added reagent does not influence the dyeing effect on melanin in pathological tissues. The kit containing the ferrous sulfate reagent is used for dyeing melanin, and more accurate diagnosis basis can be provided for clinicians to diagnose melanin lesions such as skin melanoma, neuromelanoma, pseudomelanosis pigment and the like.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a ferrous sulfate reagent for melanin dyeing and a kit.
Background
Melanin belongs to a non-limbic endogenous pigment, and is a group of pigments ranging in color from light brown to black. This pigment is usually found in the substantia nigra and hair follicles of the skin, eyes, brain. In pathological conditions, melanin deposits may be present throughout the body. Melanoma is often considered when it is confirmed in tumor cells suspected of being present in melanoma, particularly undifferentiated tumor tissue, or within lymph nodes. Melanin staining is used in the diagnosis of melanopathies such as cutaneous melanoma, neuromelanoma, pseudomelanosis pigment, and the like.
The ferrous sulfate dyeing method is a common melanin dyeing method, and is a ferrous ion adsorption method, wherein the melanin can absorb ferrous ions, namely the ferrous ions are adsorbed on the melanin to form a melanin ferrous complex, and then potassium ferricyanide is combined with the ferrous ions to generate iron ferricyanide, namely the Tengern blue reaction in an acid environment.
The existing ferrous sulfate solution is required to be prepared when used for melanin dyeing, yellow precipitate can be generated after the solution is placed for about 2 hours after the preparation, the solution dyeing is invalid, and if a little hydrochloric acid is added into the prepared ferrous sulfate solution, the oxidation of ferrous sulfate can be slowed down, but the dyeing capacity of ferrous sulfate is almost reduced by more than 50%, so that the preservation and the application of a dyeing reagent for ferrous sulfate are greatly limited. Therefore, how to effectively protect the dyeing performance of the ferrous sulfate dyeing reagent while prolonging the storage time of the ferrous sulfate dyeing reagent is a difficult problem to be solved urgently.
Disclosure of Invention
The invention aims to provide an improved ferrous sulfate reagent for melanin dyeing.
Another object of the present invention is to provide a kit comprising the above ferrous sulfate reagent, and a method for melanin staining using the kit.
The third purpose of the invention is to provide the application of the sulfuric acid subunit reagent or the kit in the staining of pathological tissues of the melanotic lesion.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a ferrous sulfate reagent for melanin dyeing, which is prepared by the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adding a non-oxidizing acid to adjust the pH value to 2.0-2.5, and adding a dyeing protective agent, wherein the protective agent is 0.1-1% of gelatin.
Preferably, the non-oxidizing acid is hydrochloric acid, dilute sulfuric acid or phosphoric acid.
Preferably, the protective agent is 0.5 mass percent of gelatin.
The invention provides a kit for melanin dyeing, which comprises the ferrous sulfate reagent.
Specifically, the kit for melanin dyeing provided by the invention mainly comprises a ferrous sulfate reagent, potassium ferricyanide acetic acid solution, Van Gieseon (VG) dyeing solution and glacial acetic acid solution.
The preparation method of the potassium ferricyanide acetic acid solution comprises the following steps: 1g of potassium ferricyanide was dissolved in 99mL of distilled water, and 1mL of glacial acetic acid was added.
The Van Gieseon (VG) staining solution was diluted with saturated picric acid: the aqueous solution of acid fuchsin was prepared at a ratio of 9: 1.
The glacial acetic acid solution is a 1% glacial acetic acid aqueous solution, and the preparation method comprises the following steps: 1mL of glacial acetic acid was measured and added to 99mL of distilled water.
The invention also provides a method for performing melanin dyeing by using the kit, which comprises the following steps:
(1) the slices are dewaxed conventionally to be hydrated;
(2) treating with ferrous sulfate reagent for 1 hr;
(3) washing with distilled water for 3 times, each for 3-5 min;
(4) treating with potassium ferricyanide acetic acid solution for 30 minutes;
(5) slightly washing with glacial acetic acid solution for 2-3 seconds;
(6) VG staining solution is used for comparative staining, about 1 minute, and the solution is slightly washed by tap water;
(7) slightly washing with 95% alcohol, and dehydrating with anhydrous ethanol;
(8) xylene transparent, sealing with neutral gum, and examining under microscope.
The invention also provides the application of the ferrous sulfate reagent or the kit in the staining of pathological tissues of the melanotic lesion.
The ferrous sulfate reagent or the kit is used for pathological tissue staining of the melanopathy, so that a diagnosis basis can be provided for a clinician to diagnose the melanopathy, and the melanopathy comprises diseases such as skin melanoma, neuromelanoma, pseudomelanosis pigment and the like.
The invention has the following beneficial effects:
1) different from the traditional ferrous sulfate dyeing reagent which is required to be used at first, the ferrous sulfate reagent provided by the invention has the advantages that the pH value is adjusted by adding the non-oxidizing acid into the ferrous sulfate solution to ensure that the pH value is 2.0-2.5, and 0.1-1% of gelatin is added as a dyeing protective agent, so that the ferrous sulfate can be fully protected from being oxidized, and the dyeing effect of melanin is not weakened. The reagent is simple to prepare, the components of the dyeing protective agent are simple and easy to obtain, the protection effect on the ferrous sulfate dyeing effect is good, the added acid and the protective agent do not influence the dyeing effect on melanin in pathological tissues, and the dyeing performance of the ferrous sulfate dyeing reagent is effectively protected while the storage time of the ferrous sulfate dyeing reagent is prolonged.
2) The kit provided by the invention can clearly dye melanin in tissues into green to dark green particles with different sizes, collagen fibers are red, muscle fibers are yellow, the dyeing result is stable, the quality guarantee period of reagents is long, the effective period of the unopened kit can reach 1 year, and great convenience is provided for the clinical popularization and application of a melanin ferrous sulfate dyeing detection technology.
Drawings
FIG. 1 is a graph showing the change of time after a ferrous sulfate solution prepared in example 1 is adjusted to pH 2.1 by hydrochloric acid and gelatin with a mass fraction of 0.1% is added as a protective agent;
in the figure, the solution remained clear after one month at room temperature (right).
FIG. 2 is a graph showing the change of pH over time of a ferrous sulfate solution prepared in example 2 by adjusting pH to 2.5 with hydrochloric acid and adding gelatin as a protective agent in an amount of 0.5% by mass;
in the figure, the solution remained clear after one month at room temperature (right).
FIG. 3 is a graph showing the change of time after the configuration of ferrous sulfate solution prepared in example 3 by adjusting pH to 2.5 with dilute sulfuric acid and adding gelatin as a protective agent with a mass fraction of 0.5%;
in the figure, the solution remained clear after one month at room temperature (right).
FIG. 4 is a graph showing the change of time of a ferrous sulfate solution prepared in example 4 by adjusting pH to 2.5 with phosphoric acid and adding gelatin as a protective agent with a mass fraction of 0.5%;
in the figure, the solution remained clear after one month at room temperature (right)
Fig. 5 is a graph showing the change of time after the configuration provided in example 5, in which a ferrous sulfate solution adjusted to pH 2.0 using hydrochloric acid and gelatin as a protective agent was added in a mass fraction of 1%.
In the figure, the solution remained clear after one month at room temperature (right).
FIG. 6 is a photograph of the 2.5% ferrous sulfate solution provided in comparative example 1 without the addition of acid or stain protectant immediately after formulation (left) and after 4-5 hours at room temperature (right);
in the figure, the solution appeared cloudy after 4-5 hours at room temperature (right).
FIG. 7 is a photograph of a 2.5% ferrous sulfate solution provided in comparative example 2 immediately after preparation (left) and after being left at room temperature for 1 month (right) by adjusting the pH to 2.2 with hydrochloric acid;
in the figure, the solution remained clear after 1 month at room temperature (right).
FIG. 8 is a graph showing the result of staining in example 6;
FIG. 9 is a graph showing the result of staining in example 7;
FIG. 10 is a graph showing the result of staining in example 8;
FIG. 11 is a graph showing the result of staining in example 9;
FIG. 12 is a graph showing the result of dyeing in example 10;
FIG. 13 is a graph showing the dyeing results of comparative example 3;
FIG. 14 is a graph showing the results of staining with the kit prepared freshly with the stain provided in example 2;
FIG. 15 is a graph showing the results of staining after 1 month of standing after the dye solution of the kit provided in example 2 was prepared;
FIG. 16 is a graph showing the results of staining after 3 months of standing after the dye solution of the kit provided in example 2 was prepared;
FIG. 17 is a graph showing the results of staining after 6 months of standing after the dye solution of the kit provided in example 2 was prepared;
FIG. 18 is a graph showing the results of staining after 12 months of standing after the dye solution of the kit provided in example 2 was prepared.
Detailed Description
The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto:
example 1
The embodiment provides an improved ferrous sulfate reagent for melanin dyeing, which is prepared by the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adjusting the pH value of the solution to 2.1 by hydrochloric acid, weighing 0.1g of gelatin, adding the gelatin into the solution, fully stirring to dissolve the gelatin, and fixing the volume of the solution to 100 mL.
The time course of the solution after preparation is shown in figure 1, and the solution is still clear after the ferrous sulfate reagent is placed at room temperature for 1 month (right).
Example 2
The embodiment provides an improved ferrous sulfate reagent for melanin dyeing, which is prepared by the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adjusting the pH value of the solution to 2.5 by hydrochloric acid, weighing 0.5g of gelatin, adding the gelatin into the solution, fully stirring to dissolve the gelatin, and fixing the volume of the solution to 100 mL.
The time course of the solution after preparation is shown in fig. 2, and the solution is still clear after the ferrous sulfate reagent is placed at room temperature for 1 month (right).
Example 3
The embodiment provides an improved ferrous sulfate reagent for melanin dyeing, which is prepared by the following steps: 2.5g ferrous sulfate is dissolved in distilled water, the pH value of the solution is adjusted to 2.5 by dilute sulfuric acid, 0.5g gelatin is weighed and added into the solution, the solution is fully stirred to be dissolved, and the volume of the solution is adjusted to 100 mL.
The time course of the solution after preparation is shown in fig. 3, and the solution is still clear after the ferrous sulfate reagent is placed at room temperature for 1 month (right).
Example 4
The embodiment provides an improved ferrous sulfate reagent for melanin dyeing, which is prepared by the following steps: dissolving 2.5g ferrous sulfate in distilled water, adjusting the pH value of the solution to 2.5 by using phosphoric acid, weighing 0.5 gelatin, adding the gelatin into the solution, fully stirring to dissolve the gelatin, and fixing the volume of the solution to 100 mL.
The time course of the solution after preparation is shown in fig. 4, and the solution is still clear after the ferrous sulfate reagent is placed at room temperature for 1 month (right).
Example 5
The embodiment provides an improved ferrous sulfate reagent for melanin dyeing, which is prepared by the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adjusting the pH value of the solution to 2.0 by hydrochloric acid, weighing 1.0g of gelatin, adding the gelatin into the solution, fully stirring to dissolve the gelatin, and fixing the volume of the solution to 100 mL.
The time course of the solution after preparation is shown in fig. 5, and the solution is still clear after the ferrous sulfate reagent is placed at room temperature for 1 month (right).
Comparative example 1
The embodiment is a traditional ferrous sulfate reagent, and the preparation method comprises the following steps: 2.5g of ferrous sulfate was dissolved in 100mL of distilled water, and the resulting solution was sufficiently stirred to dissolve it. The change of the solution with time after preparation is shown in figure 6, and turbidity appears after the solution is placed at room temperature for 4-5 hours without adjusting the pH value by acid and adding a 2.5 percent ferrous sulfate solution of a dyeing protective agent.
Comparative example 2
The preparation method of the ferrous sulfate solution of the embodiment comprises the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adjusting the pH value to 2.2 by hydrochloric acid, then metering to 100mL, and fully stirring to dissolve the ferrous sulfate. The solution was dispensed and the time course of the solution was as shown in FIG. 7, and the ferrous sulfate solution remained clear after 1 month at room temperature (right).
Example 6
The embodiment provides a kit for melanin dyeing, which comprises the ferrous sulfate reagent described in embodiment 1, and further comprises potassium ferricyanide acetic acid solution, VG dyeing solution and glacial acetic acid solution.
The preparation method of the potassium ferricyanide acetic acid solution comprises the following steps: 1g of potassium ferricyanide was dissolved in 99mL of distilled water, and 1mL of glacial acetic acid was added.
The VG staining solution is prepared by mixing saturated picric acid: the aqueous solution of acid fuchsin was prepared at a ratio of 9: 1.
The glacial acetic acid solution is a 1% glacial acetic acid aqueous solution, and the preparation method comprises the following steps: 1mL of glacial acetic acid was measured and added to 99mL of distilled water.
Example 7
The embodiment provides a kit for melanin dyeing, which comprises the ferrous sulfate reagent described in embodiment 2, and further comprises potassium ferricyanide acetic acid solution, VG dyeing solution and glacial acetic acid solution, wherein the configuration of the potassium ferricyanide acetic acid solution, the VG dyeing solution and the glacial acetic acid solution is the same as that in embodiment 7.
Example 8
The embodiment provides a kit for melanin dyeing, which comprises the ferrous sulfate reagent described in embodiment 3, and further comprises potassium ferricyanide acetic acid solution, VG dyeing solution and glacial acetic acid solution, wherein the configuration of the potassium ferricyanide acetic acid solution, the VG dyeing solution and the glacial acetic acid solution is the same as that in embodiment 7.
Example 9
The embodiment provides a kit for melanin dyeing, which comprises the ferrous sulfate reagent described in embodiment 4, and further comprises potassium ferricyanide acetic acid solution, VG dyeing solution and glacial acetic acid solution, wherein the configuration of the potassium ferricyanide acetic acid solution, the VG dyeing solution and the glacial acetic acid solution is the same as that in embodiment 7.
Example 10
The embodiment provides a kit for melanin dyeing, which comprises the ferrous sulfate reagent described in embodiment 5, and further comprises potassium ferricyanide acetic acid solution, VG dyeing solution and glacial acetic acid solution, wherein the configuration of the potassium ferricyanide acetic acid solution, the VG dyeing solution and the glacial acetic acid solution is the same as that in embodiment 7.
Comparative example 3
This example provides a kit for melanin staining, which includes the ferrous sulfate solution described in comparative example 2, and further includes potassium ferricyanide acetic acid solution, VG staining solution, and glacial acetic acid solution, and the configuration method thereof is the same as that in example 3.
Test example 1
In this test example, the immunohistochemical kit described in examples 6 to 10 and comparative example 3 was applied to the melanin staining test of pathological tissues. The types of pathological tissues and the number of cases for melanin staining test are shown in the following table:
| tissue type | Number of examples |
| Skin(s) | 5 |
| Aversion to black | 2 |
| Lung (lung) | 2 |
| Sausage | 3 |
| Neuroma of nerve | 3 |
The specific dyeing steps are as follows:
(1) the slices are dewaxed conventionally to be hydrated;
(2) treating with ferrous sulfate reagent for 1 hr;
(3) washing with distilled water for 3 times, each for 3-5 min;
(4) treating with potassium ferricyanide acetic acid solution for 30 minutes;
(5) slightly washing with glacial acetic acid solution for 2-3 seconds;
(6) VG staining solution is used for comparative staining, about 1 minute, and the solution is slightly washed by tap water;
(7) slightly washing with 95% alcohol, and dehydrating with anhydrous ethanol;
(8) xylene transparent, sealing with neutral gum, and examining under microscope.
The melanin absorbs ferrous ions in the solution to form a melanin ferrous compound, and then potassium ferricyanide is combined with the ferrous ions to generate potassium ferricyanide ferrous in an acidic environment, namely the Turnbull's blue reaction. The Van gieseon staining solution is related to the penetration of tissues by the size of anionic dye molecules, and the final staining result is that muscle fibers are dyed yellow by saturated picric acid, and collagen fibers are dyed red again by acid. Therefore, as a result of the dyeing, melanin should be dyed into green to dark green particles with different sizes, collagen fibers are red, and muscle fibers are yellow.
As can be seen from comparison of fig. 1 to 7, the existing ferrous sulfate solution must be prepared when used for melanin dyeing, yellow precipitates are generated after the solution is prepared and placed for about 2 hours, which results in ineffective solution dyeing (comparative example 1, fig. 6), and if a small amount of hydrochloric acid is added to the prepared ferrous sulfate solution, which can slow down ferrous sulfate oxidation (comparative example 2, fig. 7), the ferrous sulfate reagents provided in comparative example 2 and examples 1 to 5 of the present invention can be prepared and placed for one month, and then the dyeing ability of ferrous sulfate is greatly reduced (> 50%), which greatly limits the preservation and application of the dyeing reagent for ferrous sulfate (see fig. 13).
As can be seen from fig. 8-12, the reagent kit comprising the ferrous sulfate reagent provided by the present invention can dye melanin into green to dark green particles with different sizes, the collagen fibers are red, the muscle fibers are yellow, and the dyeing results of different treatments in different embodiments are slightly different. The result shows that the pH value is adjusted by adding the non-oxidizing acid into the ferrous sulfate solution to be 2.0-2.5, and 0.1-1% of gelatin is added to be used as a dyeing protective agent, so that the ferrous sulfate can be fully protected from being oxidized, the dyeing effect of melanin in pathological tissues is not influenced by the added acid and the protective agent, and the dyeing performance of the ferrous sulfate dyeing agent is effectively protected while the storage time of the ferrous sulfate dyeing agent is prolonged.
Test example 2
In this test example, the melanin staining kit provided in example 2 is used as an example to detect the timeliness of the kit and evaluate the shelf life of the kit. The pathological tissues, staining method and procedure for melanin staining detection were the same as in test example 1.
As can be seen from fig. 14 to 18, the kit comprising the ferrous sulfate reagent provided by the invention can clearly dye melanin in tissues into green to dark green particles with different sizes, collagen fibers are red, muscle fibers are yellow, the dyeing result is stable, the quality guarantee period of the reagent is long, the effective period of the unopened kit can reach 1 year, and great convenience is provided for clinical popularization and application of a ferrous sulfate dyeing detection technology for melanin.
Finally, the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same. The basic principles and the main features of the invention have been described above with specific embodiments, on the basis of which some modifications or alterations can be made without departing from the essence of the corresponding technical solution.
Claims (9)
1. A ferrous sulfate reagent for melanin dyeing is characterized in that the preparation method of the ferrous sulfate reagent comprises the following steps: dissolving 2.5g of ferrous sulfate in distilled water, adding non-oxidizing acid to adjust the pH value to 2.0-2.5, adding a dyeing protective agent, fully stirring to dissolve the ferrous sulfate, and fixing the volume of the solution to 100 mL; the dyeing protective agent is gelatin with the mass fraction of 0.1-1%; the non-oxidizing acid is hydrochloric acid, dilute sulfuric acid or phosphoric acid.
2. Use of the ferrous sulfate reagent of claim 1 for staining pathological tissues of melanotic lesions.
3. A kit for melanin staining comprising the ferrous sulfate reagent of claim 1.
4. The kit according to claim 3, further comprising potassium ferricyanide acetic acid solution, wherein the potassium ferricyanide acetic acid solution is prepared by a method comprising: 1g of potassium ferricyanide was dissolved in 99mL of distilled water, and 1mL of glacial acetic acid was added.
5. The kit of claim 3, further comprising a Van Gieseon (VG) staining solution, wherein the Van Gieseon (VG) staining solution is formulated in a ratio of saturated picric acid to 1% aqueous acid fuchsin = 9: 1.
6. The kit of claim 3, further comprising glacial acetic acid, wherein the glacial acetic acid is a 1% glacial acetic acid aqueous solution.
7. The kit according to claim 3, wherein the method of using the kit comprises the steps of:
(1) the slices are dewaxed conventionally to be hydrated;
(2) treating with ferrous sulfate reagent for 1 hr;
(3) washing with distilled water for 3 times, each for 3-5 min;
(4) treating with potassium ferricyanide acetic acid solution for 30 minutes;
(5) slightly washing with glacial acetic acid solution for 2-3 seconds;
(6) comparative staining with Van Gieseon (VG) staining solution, about 1 minute, slightly washed with tap water;
(7) slightly washing with 95% alcohol, and dehydrating with anhydrous ethanol;
(8) xylene transparent, sealing with neutral gum, and examining under microscope.
8. Use of a kit according to any one of claims 3 to 7 for staining of melanotic pathologies.
9. The use of claim 8, wherein the melanopathy comprises cutaneous melanoma, neuromelanoma and pseudomelanosis pigment.
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