CN108801741A - Haematoxylin for immunohistochemical staining redyes liquid and colouring method - Google Patents
Haematoxylin for immunohistochemical staining redyes liquid and colouring method Download PDFInfo
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- 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 title claims abstract description 68
- 239000007788 liquid Substances 0.000 title claims abstract description 60
- 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 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000011532 immunohistochemical staining Methods 0.000 title claims abstract description 14
- 238000004040 coloring Methods 0.000 title abstract description 14
- 102000036639 antigens Human genes 0.000 claims abstract description 26
- 108091007433 antigens Proteins 0.000 claims abstract description 26
- 238000004043 dyeing Methods 0.000 claims abstract description 26
- 239000000427 antigen Substances 0.000 claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 235000019441 ethanol Nutrition 0.000 claims description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052751 metal Inorganic materials 0.000 claims description 8
- 239000002184 metal Substances 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 7
- 239000007800 oxidant agent Substances 0.000 claims description 7
- 230000001590 oxidative effect Effects 0.000 claims description 7
- 229940037003 alum Drugs 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 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 description 4
- 230000006698 induction Effects 0.000 claims description 4
- 239000003223 protective agent Substances 0.000 claims description 4
- 229910000474 mercury oxide Inorganic materials 0.000 claims description 3
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 claims description 3
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical group CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 claims description 3
- 239000011697 sodium iodate Substances 0.000 claims description 3
- 229940032753 sodium iodate Drugs 0.000 claims description 3
- 235000015281 sodium iodate Nutrition 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- 230000036571 hydration Effects 0.000 claims 1
- 238000006703 hydration reaction Methods 0.000 claims 1
- HFFLGKNGCAIQMO-UHFFFAOYSA-N trichloroacetaldehyde Chemical compound ClC(Cl)(Cl)C=O HFFLGKNGCAIQMO-UHFFFAOYSA-N 0.000 claims 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 238000003364 immunohistochemistry Methods 0.000 description 10
- 238000010438 heat treatment Methods 0.000 description 9
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000002378 acidificating effect Effects 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 230000008439 repair process Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 206010061534 Oesophageal squamous cell carcinoma Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 208000036765 Squamous cell carcinoma of the esophagus Diseases 0.000 description 3
- 208000009453 Thyroid Nodule Diseases 0.000 description 3
- 208000024770 Thyroid neoplasm Diseases 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 208000007276 esophageal squamous cell carcinoma Diseases 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229940010048 aluminum sulfate Drugs 0.000 description 2
- 239000000981 basic dye Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 2
- 239000008236 heating water Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241001062009 Indigofera Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000006701 autoxidation reaction Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 208000014617 hemorrhoid Diseases 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—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
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- Health & Medical Sciences (AREA)
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- 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)
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Abstract
This application discloses a kind of haematoxylins for immunohistochemical staining to redye liquid and colouring method.It is Mayer ' s haematoxylin dyeing liquid that the haematoxylin, which redyes liquid, wherein the pH value that the haematoxylin redyes liquid is 2.4~2.8.The haematoxylin of the present invention redyes liquid can obtain the high coloring of color contrast after antigen retrieval step.
Description
Technical field
The present invention relates to immunohistochemistry microtomy fields, and in particular to the haematoxylin in prepared by immunohistochemistry slice is redyed
Liquid and colouring method.
Background technology
Immunohistochemistry is " goldstandard " technology in pathology department's lesion detection, wherein redying the counterstain effect of liquid to immune group
The observation for changing signal is most important.Haematoxylin redye liquid be it is most common in immunohistochemistry redye one of liquid, it by with cell
Acidic materials in core in conjunction with and so that nucleus bluish violet.Biological tissue is fixed, by aldehydes reagent after paraffin embedding, is exempting from
During epidemic disease histochemical staining, most of primary antibodies need to carry out tissue antigen recovery.Tissue samples by aldehydes reagent fixation after,
Antigen protein in tissue generates crosslinking with aldehyde radical, so that its antigenic determinant is closed, influences the combination of antibody and determined antigen.
Antigen thermal induction reparation is to utilize high temperature or high pressure tactics, coordinates certain buffer solution, opens the crosslinking of tissue antigen and aldehyde radical,
Tissue antigen is set fully to expose, to improve the recall rate of tissue antigen.And during high temperature or high temperature and pressure, cell can be destroyed
Acid structure in core makes haematoxylin redye exception occur.
Therefore, haematoxylin redyes counterstain effect of the liquid after immunohistochemistry antigen retrieval and unsatisfactory.After redying
Nucleus blue-green, rather than conventional bluish violet.And blue-green is not very clear under the background of taupe, to shadow
Ring the observation of immunohistochemistry positive signal.
Invention content
In view of this, can not be influenced by immunohistochemistry antigen retrieval the purpose of the present invention is to provide a kind of, and pass through
Its normal bluish violet is presented in haematoxylin haematoxylin after redying redyes liquid and colouring method, so as to prepare contrast
Height, dyeing are clearly sliced.
For this purpose, the first aspect of the present invention provides a kind of bush of the immunohistochemical staining for biological tissue section
Element redyes liquid.It is Mayer ' s haematoxylin dyeing liquid that the haematoxylin, which redyes liquid, wherein the pH value that the haematoxylin redyes liquid is
2.4~2.8.Preferably, the pH value that the present invention redyes liquid is 2.5~2.8, more preferably 2.5~2.7, most preferably 2.6.
The haematoxylin of the present invention redyes liquid and may include metal salt mordant, hematoxylin, oxidant, protective agent, and optional
Organic solvent.
In a particular embodiment, the metal salt mordant can be selected from alum, alum and aluminum sulfate
It is one or more.The concentration of the metal salt mordant can be 9~120g/L, preferably 9~85g/L, further preferably 9~
54g/L。
The hematoxylic concentration can be 1~7g/L, preferably 3~5g/L, more preferably 5g/L.
The oxidant can be sodium iodate and/or mercury oxide.The concentration of the oxidant can be 0.1~1.2g/L, preferably
For 0.3~1.0g/L, more preferably 0.3~0.7g/L.
The protective agent can be glycerine and/or chloraldurate.The concentration of glycerine can be 180~420mL/L, preferably
180~300mL/L, more preferably 250~300mL/L.The concentration of chloraldurate can be 10~100g/L, preferably 30~90g/
L, more preferably 50~70g/L.
The organic solvent can be ethylene glycol and/or ethyl alcohol.The concentration of the organic solvent can be 0~70mL/L, preferably
For 35~40mL/L.Organic solvent is therefore can not also to be included in the haematoxylin for helping hematoxylin to dissolve and redye
In liquid.The haematoxylin of various concentration will present different pH in the case where not adding any soda acid, and hydrochloric acid, ice vinegar can be used
Acid, citric acid or sodium hydroxide solution are adjusted.
According to the second aspect of the invention, a kind of immunohistochemical staining method of biological tissue section, the method are provided
Including：Above-mentioned haematoxylin using the present invention is redyed liquid and is redyed.
According to specific embodiment, described redye carries out after antigen retrieval step.
Antigen retrieval step in the method for the present invention can be used conventional any mode and carry out, such as with thermal induction antigen
Repair mode carries out, such as any one heating means that can be used in hyperbaric heating, heating water bath and microwave heating are repaiied
It is multiple.
Preferably, the counterstaining step carries out 0.5~2 minute.
The counterstaining step can carry out under ambient temperature.
Biological tissue of the present invention is preferably mammal, especially the tissue of human body, especially tumor tissues, breath
Meat, hemorrhoid, calculus, tubercle etc..
The haematoxylin of the present invention redyes liquid based on traditional Mayer haematoxylin dyeing liquid, by accurately adjusting its pH
Value astoundingly obtains the bluish violet coloring of color clear in the range of 2.4~2.8, can make coloured portions with
The background color of taupe generates striking contrast.
The haematoxylin of the present invention redyes ingredient and outfit that liquid does not change traditional dyeing liquid, is adjusted merely by its pH value just
Said effect is obtained, thus can be easily adaptable in the method for conventional immunohistochemical staining.Moreover, using the present invention
Haematoxylin redyes the dyeing of liquid, and each step that preparation method is sliced to conventional immunohistochemistry is not required to make a change, and suitable
For various antigen retrieval methods, the especially various methods using high temperature reparation can obtain bright bluish violet dyeing effect
Fruit.
Description of the drawings
Fig. 1 be using identical haematoxylin redye formula of liquid under different pH value to various tumor tissue sections dye after
Microscope photo, be shown in wherein A thyroid nodule tissue slice respectively pH be 2.3,2.6 and 2.9 (by it is left extremely
It is right) redye the effect redyed in liquid；The slice for shown in B being esophageal squamous cell carcinoma tissue is respectively 2.3,2.6 and 2.9 in pH
(from left to right) redye the effect redyed in liquid；It is respectively 2.3,2.6 in pH with being the swell slice of object tissue of lung shown in C
The effect redyed in liquid is redyed with 2.9 (from left to right).
Specific implementation mode
Below in conjunction with the attached drawing in embodiment of the present invention, the technical solution in embodiment of the present invention is carried out clear
Chu is fully described by, it is clear that and described embodiment is only a part of embodiment of the present invention, rather than whole
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art are without creative efforts
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has and is led with belonging to the present invention
The identical meaning of general understanding of field technique personnel.If there are contradiction, this specification is preferential.
Mayer ' s haematoxylin dyeing liquid, also referred to as MayerShi haematoxylins, Mayer ' s haematoxylins, Mayor haematoxylins etc..
Mayer ' s haematoxylin dyeing liquid is one kind of haematoxylin dyeing liquid, is often used and redyes liquid as immunohistochemistry.Its energy and cell
Acidic materials in core combine, and nucleus is made bluish violet.
In haematoxylin dyeing liquid, hematoxylin is basic dyes, since dissolubility is poor in water for it, therefore uses wine more
The molten reagent dissolving of the alcohol such as essence, ethylene glycol, needed after the dissolving of hematoxylin dyestuff under the action of metal salt mordant could with it is intracellular
Acidic materials combine, common metal salt mordant includes alum, alum, aluminum sulfate etc..Hematoxylin is in the solution
Colorability it is closely bound up by degree of oxidation with it, can autoxidation in air, but rate is slower, quick to reach
The purpose used, it is often necessary to oxidant, such as mercury oxide, sodium iodate are added in haematoxylin dyeing liquid.In addition, with bush
The degree of oxidation of plain dyeing liquor is gradually deepened, and can be accompanied by crystal and be precipitated, to achieve the purpose that protect reagent, it is often necessary to be added
The reagents such as glycerine, chloraldurate.
The inventors discovered that in the immunohistochemical staining of histotomy, the slice color of liquid dyeing is redyed with haematoxylin
It does not show bluish violet common in routine hematoxylin dyeing, but shows blue-green.And such color and immunohistochemistry
The degradation in contrast of the taupe background of the slice of dyeing, keeps coloring not ideal enough.
Has the largely research about haematoxylin dyeing liquid.These researchs, which focus mostly on, is adjusting dyeing liquor ingredient or ingredient
Proportioning.However, the inventors discovered that, change dyeing liquor ingredient or composition proportion and not can effectively improve after antibody repairs step
Counterstain effect.
Surprisingly, the inventors discovered that, it is bright that the minor change of haematoxylin dyeing liquid pH value causes coloring to have
Aobvious difference.When wherein slightly above conventional 2.1~2.3 pH value, best coloring is obtained.This is mainly due to haematoxylin
Dyeing liquor is basic dye, is influenced by environmental pH to the coloring of nucleus, and antigen retrieval step can destroy nucleus
Interior acidic materials environment or structure, this change can influence coloring of the haematoxylin to nucleus.
The advantages of by the following specific examples further illustrate the invention.
Material and method
The clinical sample for obtaining the swollen object of thyroid nodule, esophageal squamous cell carcinoma and lung is sliced for making.
4 μm of ultra-thin sections will be processed into using slicer (coming card, RM2235) after clinical sample paraffin embedding.By following step
Suddenly it is dyed.
Dewaxing：Histotomy is dipped in dimethylbenzene I and dimethylbenzene II each 3-5 minutes.
Rehydration：It immerses successively in 100% ethyl alcohol I, 100% ethyl alcohol II, 95% ethyl alcohol, 85% ethyl alcohol and 75% ethyl alcohol, respectively
1-3 minutes, then originally flow was washed 1 minute~2 minutes.
Antigen retrieval：Three kinds of repair mode selections are a kind of below.
Hyperbaric heating reparation：Appropriate EDTA or citric acid antigen are repaired liquid, and (Foochow steps the limited public affairs of neoformation technological development
Department) be put into and be heated to boiling in pressure cooker after, histotomy is put into antigen retrieval buffers.Continue in 500W-1500W after closed
Heating.Then timing 1 minute~2 minutes after pressure valve starts jet stop heating, after cooled to room temperature, taking-up group
Knit slice.
Heating water bath reparation：Appropriate EDTA is added in a reservoir or citric acid antigen repairs liquid (Foochow neoformation technology advanced in years
Development corporation, Ltd.), after being heated to boiling, histotomy is put into antigen retrieval buffers, continues heating 15-25 minutes, preferably
After twenty minutes, stop heating, after cooled to room temperature, take out histotomy.
Microwave heating reparation：It is put into appropriate EDTA in a reservoir or citric acid antigen repairs liquid (Foochow neoformation technology advanced in years
Development corporation, Ltd.), it is put into micro-wave oven, high power is heated to boiling, and histotomy is put into antigen retrieval buffers, middle Gao Gong
Rate continues microwave 7-13 minutes, preferably 10 minutes, after cooled to room temperature, takes out histotomy.
Block endogenous peroxydase：Peroxidase blocking reagent incubation at room temperature is added in slice after antigen retrieval；
Add primary antibody：After primary antibody incubation is added in histotomy, PBS solution is rinsed 2~5 minutes, is flushed three times.
Add secondary antibody：After secondary antibody incubation is added in histotomy, PBS solution is rinsed 2~5 minutes, is flushed three times.
Colour developing：PBS solution is removed, the colour developing of DAB developing solutions is added.
It redyes：Histotomy immersion haematoxylin is redyed in liquid, is dyed 30 seconds to 2 minutes, tap water flowing water rinses 2 points
Clock；
Color separation：(1% ethanol solution hydrochloride need to be prepared voluntarily within 1~3 second for color separation in 0.5~1% acidic alcohol：
99.0mL75% ethyl alcohol adds 1.0mL hydrochloric acid).
Return indigo plant：Originally flow is washed 7 minutes~10 minutes；
Dehydration：75% ethyl alcohol, 85% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol I and absolute ethyl alcohol II are immersed successively, and each 1-3 divides
Clock；
It is transparent：It is put into dimethylbenzene I and dimethylbenzene II each 3-5 minutes；
Neutral gum mounting, microscope (Olympus, BX53) observation.
Examples 1 to 5 and comparative example 1~3
Haematoxylin, which is prepared, according to formula as below redyes liquid：
The slice for taking the clinical sample of the swollen object of the thyroid nodule, esophageal squamous cell carcinoma and lung prepared respectively, according to above-mentioned side
Optimum condition in method carries out immunohistochemical staining, and condition is as shown in table 1 below.Because of the isoelectric point of acidic materials in nucleus
It is 3.0~3.3, so theoretically when pH value is less than 3.0, haematoxylin could colour nucleus.Therefore, the pH value of liquid is redyed
It is set separately between 2.1~2.9.
Table 1：
The tone of haematoxylin dyeing during visual observations are sliced after dyeing, and calculate the ratio of normal color and luster (bluish violet) slice
Example.As a result as shown in table 2 below.
Table 2：
| PH value | Main tone | Bluish violet ratio | Coloring degree | |
| Comparative example 1 | 2.1 | Blue-green | 50% | Fully |
| Comparative example 2 | 2.3 | Blue-green | 59% | Fully |
| Embodiment 1 | 2.4 | Blue-green/bluish violet | 79% | Fully |
| Embodiment 2 | 2.5 | Bluish violet | 96% | Fully |
| Embodiment 3 | 2.6 | Bluish violet | 100% | Fully |
| Embodiment 4 | 2.7 | Bluish violet | 100% | Fully |
| Embodiment 5 | 2.8 | Bluish violet | 100% | It is micro- shallow |
| Comparative example 3 | 2.9 | Bluish violet | 100% | It is extremely shallow |
Each histotomy for taking comparative example 2, embodiment 3 and comparative example 3 respectively, by micro- sem observation and takes pictures, photo
Comparison is referring to Fig. 1.From the above experimental result and Fig. 1 it will be clear that in immunohistochemical staining, histotomy passes through
After thermal induction is repaired, redyes liquid with the haematoxylin of different pH value and achieve visibly different counterstain effect.Wherein haematoxylin is multiple
Nucleus blue-green when dye liquor pH < 2.4, poor with the contrast effect of background color tone, and dyeing is not clear enough；When 2.4≤pH≤
Nucleus bluish violet in the overwhelming majority slice when 2.8, and comparison is very clear；And as pH > 2.8, although tone is all presented
Bluish violet, but coloring is insufficient, color is excessively shallow, and comparison is very poor.
Embodiment described above is only the preferred embodiment of the present invention, is not intended to limit the scope of the invention,
It is every the present invention inventive concept under, using equivalent structure transformation made by description of the invention and accompanying drawing content, or directly/
Other related technical areas are used in indirectly to be included in the scope of patent protection of the present invention.
Claims (12)
1. a kind of haematoxylin of immunohistochemical staining for biological tissue section redyes liquid, the haematoxylin redyes liquid and is
Mayer ' s haematoxylin dyeing liquid, wherein the pH value that the haematoxylin redyes liquid is 2.4~2.8.
2. haematoxylin according to claim 1 redyes liquid, wherein the pH value that the haematoxylin redyes liquid is 2.5~2.7.
3. haematoxylin according to claim 1 or 2 redyes liquid, wherein the haematoxylin dyeing liquid includes：Metal salt mordant,
Hematoxylin, oxidant, protective agent and optional organic solvent.
4. haematoxylin according to claim 3 redyes liquid, wherein the metal salt mordant is selected from alum, aluminum sulfate
One or more, and a concentration of 9~120g/L of the metal salt mordant, preferably 9~85g/L of ammonium and aluminum sulfate, into one
Step is preferably 9~54g/L.
5. haematoxylin according to claim 3 redyes liquid, wherein the hematoxylic a concentration of 1~7g/L, preferably 3~
5g/L, more preferably 5g/L.
6. haematoxylin according to claim 3 redyes liquid, wherein the oxidant is sodium iodate and/or mercury oxide, and institute
State a concentration of 0.1~1.2g/L of oxidant, preferably 0.3~1.0g/L, more preferably 0.3~0.7g/L.
7. haematoxylin according to claim 3 redyes liquid, wherein the protective agent is glycerine and/or chloraldurate, institute
State a concentration of 180~420mL/L of glycerine, preferably 180~300mL/L, more preferably 250~300mL/L, the hydration
A concentration of 10~100g/L of chloral, preferably 30~90g/L, more preferably 50~70g/L.
8. haematoxylin according to claim 3 redyes liquid, wherein the organic solvent is ethylene glycol and/or ethyl alcohol, it is described
A concentration of 0~70mL/L of organic solvent, preferably 35~40mL/L.
9. a kind of immunohistochemical staining method of biological tissue section, the method includes using any in such as claim 1-8
Haematoxylin described in is redyed liquid and is redyed.
10. immunohistochemical staining method according to claim 9, wherein described redye in the laggard of antigen retrieval step
Row.
11. immunohistochemical staining method according to claim 10, wherein the antigen retrieval is thermal induction antigen retrieval.
12. according to the immunohistochemical staining method of claim 9~11 any one of them biological tissue section, wherein described multiple
Step is contaminated to carry out 0.5~2 minute.
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| CN201810291344.0A CN108801741B (en) | 2018-03-30 | 2018-03-30 | Hematoxylin complex staining solution for immunohistochemical staining and staining method |
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