CN103743904A - Double-labeled immunohistochemical staining kit for micro invasive lung adenocarcinoma - Google Patents
Double-labeled immunohistochemical staining kit for micro invasive lung adenocarcinoma Download PDFInfo
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- CN103743904A CN103743904A CN201410026635.9A CN201410026635A CN103743904A CN 103743904 A CN103743904 A CN 103743904A CN 201410026635 A CN201410026635 A CN 201410026635A CN 103743904 A CN103743904 A CN 103743904A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to the technical field of immunohistochemistry and relates to a double-labeled immunohistochemical staining kit for micro invasive lung adenocarcinoma. In order to solve the problem that invasive components and non-invasive components of micro invasive lung adenocarcinoma in HE staining films are difficultly distinguished clearly, the CD34 and beta-Tubulin-III double-labeled immunohistochemical staining kit is prepared and is applied to identification of the invasive lesions and the non-invasive components in the early stage of the lung adenocarcinoma, and overdiagnosis or under-diagnosis of the lung adenocarcinoma, caused by artificial interpretation difference, can be avoided.
Description
Technical field
The invention belongs to immunohistochemistry technology field, particularly, the present invention relates to a kind of two mark immunohistochemical staining kits of the early infiltrate kitchen range that shows micro-wellability adenocarcinoma of lung.
Technical background
The therapeutic strategy difference of adenocarcinoma of lung different phase, therefore its pathological is significant to clinical treatment and prognosis.In IASLC/ATS/ERS adenocarcinoma of lung classification in 2011, the main adenocarcinoma of lung Morphological Characteristics that adopts HE dyeing carries out somatotype.But these diagnostic criteria often can run into some problems in actual applications, for example, the adherent shape gland cancer of subsiding property does not have clear and definite boundary between the two with the acinar adenocarcinoma that infiltrates growth; Focal bunch of shape growth of adherent shape gland cancer, simple nipple, papillary adenocarcinoma, micro-papillary adenocarcinoma, may be a kind of evolution continuously, but also may directly develop into micro-papillary adenocarcinoma from a bunch shape growth.Therefore, only with HE, dye to differentiate that adenocarcinoma of lung early infiltrate kitchen range acquire a certain degree of difficulty.
We find by research, and when adenocarcinoma of lung is during in the ACIS stage, tumour cell is that β-Tubulin-III is negative to be expressed, and its Stromal fibroblasts is CD34 positive expression.When tumour occurs to infiltrate, tumour cell transfers β-Tubulin-III positive expression to, and its Stromal fibroblasts transfers the negative expression of CD34 to.These two kinds of marks can clearly show the scope of adenocarcinoma of lung early infiltrate kitchen range, are conducive to the pathological diagnosis of adenocarcinoma of lung.
According to our result of study, we have designed CD34 and the two mark of β-Tubulin-III immunohistochemical staining kit, for showing that in a section micro-infiltration adenocarcinoma of lung infiltrates composition and non-infiltration composition, to avoid excessive diagnosis or the underdiagnosis of the micro-wellability adenocarcinoma of lung causing as interpretation difference because of people.
Summary of the invention
The object of the invention is to, for avoiding excessive diagnosis or the underdiagnosis of pathologist to micro-wellability adenocarcinoma of lung, provide a kind of two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung early infiltrate kitchen range.This kit dyeing course is simple and efficient, can show pathological image information abundant and that contrast property is strong same section, makes the antidiastole of micro-wellability adenocarcinoma of lung early infiltrate kitchen range more easy and definite.
A kind of two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung of the present invention, the primary antibodie of detection method is multiple antibody cocktail.
Described mixed type primary antibodie is CD34 antibody and anti-β-Tubulin-III antibody mixed liquor.
HRP and AP enzyme mark that the detection amplification system adopting is multiple genus.
The developer adopting is the two or more mixing in DAB, AP-Red, BCIP/NBT, AEC.
Described mixed type primary antibodie is at the same time simultaneous reactions of same section, and slice thickness is 3-5 μ m.
Described detection method adopts the HRP corresponding with mixed type primary antibodie and AP enzyme target mixing two to resist.
of the present invention pair of dye detecting method, comprises the following steps:
(1) histotomy is put into dry roasting 2 h of 67 ℃ of constant temperature ovens.
(2) conventional dimethylbenzene dewaxing 3 times, each 6 minutes, aquation in 100%, 100%, 95%, 85% gradient ethanol, each 3 minutes, last tap water rinsed.
(3) at the citric acid antigen retrieval liquid mesohigh of pH6.0, repair 1.5-2 minute, naturally cool to room temperature, tap water rinses, and PBS rinses 3 × 3 minutes.
(4) incubated at room 10 minutes in 3wt.% hydrogen peroxide, blocking-up endogenous peroxydase, PBS rinses 3 × 3 minutes.
(5) with Normal animal serum sealing 10 minutes, get rid of unnecessary serum, do not wash, drip and mix primary antibodie, incubated at room 1 hour, PBS rinses 3 × 3 minutes.
(6) drip mixing two and resist, incubated at room 15 minutes, PBS rinses 3 × 3 minutes.
(7) substep colour developing, monitors process color under microscope mirror, in good time cessation reaction.
(8) haematoxylin is redyed, and the sealing of water-based mounting medium, after water-based mounting medium bone dry, adds cover glass sealing with neutral gum, is beneficial to photomicrograph.
Remarkable advantage of the present invention: the two dye detecting methods of micro-wellability adenocarcinoma of lung early infiltrate kitchen range SABC, first mixed type primary antibodie and multiple genus are detected to amplification system and are applied to the antidiastole of micro-wellability adenocarcinoma of lung early infiltrate kitchen range, when not only having kept each antibody in antibody cocktail and singly having dyed outside same susceptibility and specificity, also there is staining procedure few, experimental period is short, stability is high, experimental result intuitively contrasts, and the quantity of information of bringing is singly dyed advantages of higher more than tradition.
Accompanying drawing explanation
Fig. 1 is micro-wellability adenocarcinoma of lung, and the Stromal fibroblasts of Showed by immune group result adenocarcinoma of lung non-infiltration composition (adherent shape gland cancer) is CD34 positive expression (black), but cancer cell is β-Tubulin-III feminine gender; The Stromal fibroblasts that adenocarcinoma of lung infiltrates composition (acinar adenocarcinoma) is CD34 feminine gender, but cancer cell is β-Tubulin-III strong positive, expresses (redness) (× 100).
Fig. 2 is micro-wellability adenocarcinoma of lung, and the Stromal fibroblasts of Showed by immune group result adenocarcinoma of lung non-infiltration composition (adherent shape gland cancer) is CD34 positive expression (redness), but cancer cell is β-Tubulin-III feminine gender; The Stromal fibroblasts that adenocarcinoma of lung infiltrates composition (acinar adenocarcinoma) is CD34 feminine gender, but cancer cell is β-Tubulin-III strong positive, expresses (black) (× 100).
Fig. 3 is wellability adenocarcinoma of lung, and the Stromal fibroblasts that Showed by immune group result adenocarcinoma of lung infiltrates composition (acinar adenocarcinoma) is the negative expression of CD34, but cancer cell is β-Tubulin-III strong positive, expresses (black) (× 40).
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, and following examples are in order to further illustrate the present invention, but should not be considered as limiting the present invention.
embodiment 1:
Research object is fix-paraffin-embedded micro-wellability pulmonary adenocarcinoma (pathology department of Fuzhou General Hospital of Nanjing Military Command) of formaldehyde.Immunohistochemical assay step is as follows:
(1) histotomy is put into the dry roasting 2h of 67 ℃ of constant temperature ovens.
(2) conventional dimethylbenzene dewaxing 3 times, each 6 minutes, aquation in 100%, 100%, 95%, 85% gradient ethanol, each 3 minutes, last tap water rinsed.
(3) at the citric acid antigen retrieval liquid mesohigh of pH6.0, repair 1.5-2 minute, naturally cool to room temperature, tap water rinses, and PBS rinses 3 × 3 minutes.
(4) incubated at room 10 minutes in 3% hydrogen peroxide, blocking-up endogenous peroxydase, PBS rinses 3 × 3 minutes.
(5) with Normal animal serum sealing 10 minutes, get rid of unnecessary serum, do not wash, drip CD34 and β-Tubulin-III and mix primary antibodie, mix each antibody titer in primary antibodie and be respectively CD34 1:200, β-Tubulin-III 1:400, incubated at room 1 hour, PBS rinses 3 × 3 minutes.
(6) dropping HRP, the AP enzyme target mixing two corresponding with primary antibodie resists, incubated at room 15 minutes, and PBS rinses 3 × 3 minutes.
(7) drip AP-Red nitrite ion, under room temperature, hatch 15-30min, under microscope mirror, monitor process color, in good time cessation reaction; Drip BCIP/NBT nitrite ion, under room temperature, hatch 10-30min, under microscope mirror, monitor process color, in good time cessation reaction.
(8) haematoxylin is redyed, and the sealing of water-based mounting medium, after water-based mounting medium bone dry, adds cover glass sealing with neutral gum, is beneficial to photomicrograph.
As shown in Figure 1, the Stromal fibroblasts of adenocarcinoma of lung non-infiltration composition (adherent shape gland cancer) is CD34 positive expression (black), infiltrates composition (acinar adenocarcinoma) and is β-Tubulin-III strong positive expression (redness) (× 100).
This detection method of Fig. 1 presentation of results can obviously be distinguished micro-wellability adenocarcinoma of lung early infiltrate kitchen range, by β-Tubulin-III strong positive, expresses to differentiate that this case is micro-wellability adenocarcinoma of lung.
embodiment 2:
Research object is fix-paraffin-embedded micro-wellability pulmonary adenocarcinoma (pathology department of Fuzhou General Hospital of Nanjing Military Command) of formaldehyde.Immunohistochemical assay step is as follows:
(1) histotomy is put into the dry roasting 2h of 67 ℃ of constant temperature ovens.
(2) conventional dimethylbenzene dewaxing 3 times, each 6 minutes, aquation in 100%, 100%, 95%, 85% gradient ethanol, each 3 minutes, last tap water rinsed.
(3) at the citric acid antigen retrieval liquid mesohigh of pH6.0, repair 1.5-2 minute, naturally cool to room temperature, tap water rinses, and PBS rinses 3 × 3 minutes.
(4) incubated at room 10 minutes in 3% hydrogen peroxide, blocking-up endogenous peroxydase, PBS rinses 3 × 3 minutes.
(5) with Normal animal serum sealing 10 minutes, get rid of unnecessary serum, do not wash, drip CD34 and β-Tubulin-III and mix primary antibodie, mix each antibody titer in primary antibodie and be respectively CD34 1:200, β-Tubulin-III 1:400, incubated at room 1 hour, PBS rinses 3 × 3 minutes.
(6) dropping HRP, the AP enzyme target mixing two corresponding with primary antibodie resists, incubated at room 15 minutes, and PBS rinses 3 × 3 minutes.
(7) drip AP-Red nitrite ion, under room temperature, hatch 15-30min, under microscope mirror, monitor process color, in good time cessation reaction; Drip BCIP/NBT nitrite ion, under room temperature, hatch 10-30min, under microscope mirror, monitor process color, in good time cessation reaction.
(8) haematoxylin is redyed, and the sealing of water-based mounting medium, after water-based mounting medium bone dry, adds cover glass sealing with neutral gum, is beneficial to photomicrograph.
As shown in Figure 2, the Stromal fibroblasts of adenocarcinoma of lung non-infiltration composition (adherent shape gland cancer) is CD34 positive expression (redness), infiltrates composition (acinar adenocarcinoma) and is β-Tubulin-III strong positive expression (black) (× 100).
This detection method of Fig. 2 presentation of results can obviously be distinguished micro-wellability adenocarcinoma of lung early infiltrate kitchen range, by β-Tubulin-III strong positive, expresses to differentiate that this case is micro-wellability adenocarcinoma of lung.
embodiment 3:
Research object is fix-paraffin-embedded micro-wellability pulmonary adenocarcinoma (pathology department of Fuzhou General Hospital of Nanjing Military Command) of formaldehyde.Immunohistochemical assay step is as follows:
(1) histotomy is put into the dry roasting 2h of 67 ℃ of constant temperature ovens.
(2) conventional dimethylbenzene dewaxing 3 times, each 6 minutes, aquation in 100%, 100%, 95%, 85% gradient ethanol, each 3 minutes, last tap water rinsed.
(3) at the citric acid antigen retrieval liquid mesohigh of pH6.0, repair 1.5-2 minute, naturally cool to room temperature, tap water rinses, and PBS rinses 3 × 3 minutes.
(4) incubated at room 10 minutes in 3% hydrogen peroxide, blocking-up endogenous peroxydase, PBS rinses 3 × 3 minutes.
(5) with Normal animal serum sealing 10 minutes, get rid of unnecessary serum, do not wash, drip CD34 and β-Tubulin-III and mix primary antibodie, mix each antibody titer in primary antibodie and be respectively CD34 1:200, β-Tubulin-III 1:400, incubated at room 1 hour, PBS rinses 3 × 3 minutes.
(6) dropping HRP, the AP enzyme target mixing two corresponding with primary antibodie resists, incubated at room 15 minutes, and PBS rinses 3 × 3 minutes.
(7) drip AP-Red nitrite ion, under room temperature, hatch 15-30min, under microscope mirror, monitor process color, in good time cessation reaction; Drip BCIP/NBT nitrite ion, under room temperature, hatch 10-30min, under microscope mirror, monitor process color, in good time cessation reaction.
(8) haematoxylin is redyed, and the sealing of water-based mounting medium, after water-based mounting medium bone dry, adds cover glass sealing with neutral gum, is beneficial to photomicrograph.
As shown in Figure 3, the Stromal fibroblasts that adenocarcinoma of lung infiltrates composition (acinar adenocarcinoma) is the negative expression of CD34, infiltrates composition (acinar adenocarcinoma) and is β-Tubulin-III strong positive expression (black) (× 40).
This detection method of Fig. 3 presentation of results can obviously be distinguished wellability adenocarcinoma of lung and infiltrate kitchen range, by the expression of β-Tubulin-III strong positive and negative expression of CD34, differentiates that this case is wellability adenocarcinoma of lung.
Through the research of above-mentioned three kinds of embodiments, by color contrast visually, the comparison of ImmunohistochemistryResults Results specificity, susceptibility, determines the two dye detecting methods of micro-wellability adenocarcinoma of lung early infiltrate kitchen range SABC using the method in embodiment 1 as the best.
Various reagent described in this patent, except particularly pointing out, all upper acquisitions from commercial channels.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (5)
1. for two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung, it is characterized in that: in detection method, primary antibodie is two kinds of mixed type primary antibodies.
2. a kind of two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung according to claim 1, is characterized in that: described two kinds of mixed type primary antibodies are the mixed liquor of CD34 antibody and β-Tubulin-III antibody.
3. a kind of two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung according to claim 1, is characterized in that: the developer of employing is the two or more mixing in DAB, AP-Red, BCIP/NBT, AEC.
4. a kind of two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung according to claim 1, is characterized in that: described two kinds of mixed type primary antibodies are at the same time simultaneous reactions of same section.
5. a kind of two mark immunohistochemical staining kits for micro-wellability adenocarcinoma of lung according to claim 1, is characterized in that: detection method adopts the HRP corresponding with mixed type primary antibodie and AP enzyme target mixing two to resist.
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| CN107179406A (en) * | 2017-05-27 | 2017-09-19 | 福州迈新生物技术开发有限公司 | Immunohistochemical staining kit is marked cancer embolus more |
| CN107677827A (en) * | 2017-10-09 | 2018-02-09 | 亚能生物技术（深圳）有限公司 | A kind of double transfection reagent boxes of cervical carcinoma supplementary classification diagnosis and its application |
| CN109856383A (en) * | 2019-03-05 | 2019-06-07 | 湖北泰康医疗设备有限公司 | A kind of immunochemistry staining kit for cervical carcinoma auxiliary diagnosis |
| CN110161225A (en) * | 2019-05-24 | 2019-08-23 | 中国人民解放军联勤保障部队第九0四医院 | A kind of detection cancer cell invades the immunohistochemical double-labeled kit of nerve |
| CN110850094A (en) * | 2019-11-22 | 2020-02-28 | 西安交通大学 | Immunohistochemical double-label single-staining kit and use method and application thereof |
| CN114113084A (en) * | 2021-11-11 | 2022-03-01 | 福州迈新生物技术开发有限公司 | Real-time accompanying film reading method based on digital pathological image and storage device |
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| CN107179406A (en) * | 2017-05-27 | 2017-09-19 | 福州迈新生物技术开发有限公司 | Immunohistochemical staining kit is marked cancer embolus more |
| CN107677827A (en) * | 2017-10-09 | 2018-02-09 | 亚能生物技术（深圳）有限公司 | A kind of double transfection reagent boxes of cervical carcinoma supplementary classification diagnosis and its application |
| CN109856383A (en) * | 2019-03-05 | 2019-06-07 | 湖北泰康医疗设备有限公司 | A kind of immunochemistry staining kit for cervical carcinoma auxiliary diagnosis |
| CN110161225A (en) * | 2019-05-24 | 2019-08-23 | 中国人民解放军联勤保障部队第九0四医院 | A kind of detection cancer cell invades the immunohistochemical double-labeled kit of nerve |
| CN110850094A (en) * | 2019-11-22 | 2020-02-28 | 西安交通大学 | Immunohistochemical double-label single-staining kit and use method and application thereof |
| CN114113084A (en) * | 2021-11-11 | 2022-03-01 | 福州迈新生物技术开发有限公司 | Real-time accompanying film reading method based on digital pathological image and storage device |
| WO2023082650A1 (en) * | 2021-11-11 | 2023-05-19 | 福州迈新生物技术开发有限公司 | Real-time concomitant slice reading method based on digital pathological image, and storage device |
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