CN205049576U - A dyeing kit for detecting breast cancer cell - Google Patents
A dyeing kit for detecting breast cancer cell Download PDFInfo
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- CN205049576U CN205049576U CN201520684125.0U CN201520684125U CN205049576U CN 205049576 U CN205049576 U CN 205049576U CN 201520684125 U CN201520684125 U CN 201520684125U CN 205049576 U CN205049576 U CN 205049576U
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Abstract
The utility model provides a dyeing kit for detecting breast cancer cell, this kit are one with the anti people's broad -spectrum cytokerain antibody of mouse (AE1AE3) and resist to the rabbit anti -mouse antibody of horseradish peroxidase mark is two and antily carries out the mark to breast cancer cell, dyes with the haematoxylin through diaminobenzidine (DAB), makes breast cancer cell dyeed by the specificity. The utility model discloses a method easy operation, the accuracy is good, can be used to the detection of breast cancer micrometastasis cell in breast cancer patient marrow blood and the peripheral blood sample, has important reference meaning to the prognosis of breast cancer.
Description
Technical field
The utility model belongs to technical field of biological, relates to a kind of staining kit for detecting breast cancer cell particularly.
Background technology
The evaluation index of patient with breast cancer's long-dated survival, comprise the size of primary tumor, axillary gland with or without transfer, tumor cells somatotype and the evidence etc. whether having DISTANT METASTASES IN, these indexs are very meaningful for the existence evaluation of PATIENT POPULATION, but relatively limited to the accuracy of the risk judgment of some individual patient.In addition, be had regular physical checkups by physical examination, iconography etc. inspections in the follow-up period of breast cancer, when waiting these to check and pinpoint the problems, in fact missed the best opportunity for the treatment of.At the commitment of breast cancer, considerable part patient is just had to have micrometastatic disease (the small metastatic lesion that cannot be found by general medical inspection).Previously research find that micrometastasis detects can the situation of more current follow-up examination means more early reflection patient DISTANT METASTASES IN, and micrometastasis positive patient comparatively negative patient's prognosis is poorer.1979, Heyderman detects on normal breast acinus luminal membrane by the method for immunocytochemistry (Immunocytochemistry, ICC), on the cell membrane of breast cancer cell and extensively there is the expression of EMA (EpithelialMembraneAntigen) in endochylema.Subsequently, Sloane has entered to organize the patient with breast cancer that 43 occur DISTANT METASTASES IN, add the antibody of EMA from crista iliaca collection marrow specimen, had in the marrow specimen of 8 patients the single tumour cell and a small amount of tumour cell group that there is transfer by the method Late Cambrian of ICC.These tumour cells from primary tumor depart from after, sent out to distant place target organ by lymph and/or blood circulation, hide in target organ, enter dormant state, formed Micrometastatic cell/micrometastasis stove.When under organismic internal environment change or the effect in some extraneous factor, the Micrometastatic cell of these dormancy starts again to breed, the recurrence that final formation clinical examination can find and metastatic lesion.At present, usual clinically the Micrometastatic cell being present in hematological system is called circulating tumor cell (CirculatingTumorCell, CTC); Be colonizated in then called after disseminated tumor cells (DisseminatedTumorCell, DTC) of myeloid tissue (BoneMarrow, BM).
If provide a kind of simple method to detect in marrow specimen or blood preparation whether have breast cancer Micrometastatic cell, for the Index for diagnosis of breast cancer and individualized treatment significant.
Utility model content
Utility model object: be solve problems of the prior art, the utility model provides a kind of easy and simple to handle, staining kit for detecting breast cancer cell that accuracy is good.
Technical scheme: for realizing above-mentioned technical purpose, staining kit for detecting breast cancer cell of the present utility model comprises box body, lid, microslide, the primary antibodie reagent bottle of mouse anti human wide spectrum anti-cytokeratin Ab is housed, two anti-reagent bottles of horseradish peroxidase-labeled rabbit anti-mouse antibody are housed, DAB chromogenic reagent group, DAB nitrite ion storage bottle, be equipped with and haematoxylinly redye liquid reagent bottle, the color separation liquid reagent bottle of 1% acidic alcohol is housed, the mounting liquid reagent bottle of neutral gum is housed, wherein, described box body comprises two-layer up and down, wherein, lower floor is separated into two cavitys, being respectively arranged with in two cavitys can the drawer of pull, wherein, in a described microslide cavity disposed therein, described DAB chromogenic reagent group is arranged in another cavity, described DAB chromogenic reagent group comprises the reagent bottle that DAB damping fluid is housed, the reagent bottle that DAB substrate is housed and the reagent bottle of DAB chromogen is housed, the upper strata of described box body is separated into six subregions, is respectively used to place primary antibodie reagent bottle, two anti-reagent bottles, DAB nitrite ion storage bottle, redye liquid reagent bottle, color separation liquid reagent bottle and mounting liquid reagent bottle.
Preferably, described DAB nitrite ion storage bottle is brown storage bottle.
Preferably, describedly the drawer outside of pull draw ring can be provided with.
Using method:
(1) the microslide face of surperficial plated cells is prepared;
(2) with mouse anti human wide spectrum anti-cytokeratin Ab (mouse anti human Anti-panCytokeratinantibody [AE1/AE3]) for primary antibodie, the microslide of the plated cells itself and step (1) obtained hatches 1 ~ 2h in 37 DEG C of incubators, PBS washing 3 ~ 4 times, each 3 ~ 4min;
(3) with horseradish peroxidase-labeled (HRPPolymerConjugate) rabbit anti-mouse antibody be two resist, the microslide and described two obtain step (2) resists hatches 25 ~ 30min under normal temperature, PBS washing 3 ~ 4 times, each 3 ~ 4min;
(4) microslide obtained to step (3) drips the brown nitrite ion of freshly prepared DAB, left at room temperature 5 ~ 8min, after presenting brown colouring at low power lens (10 ×) downward view inner cell, washes cessation reaction with water;
(5) with microslide 30 ~ 40s that haematoxylin counterstaining step (4) obtains, after limpid to water with tap water, with 1% acidic alcohol color separation 1s, then 1 ~ 1.5min is washed in water logging, immerse each 3 ~ 4min in 85% (v/v) ethanol, 95% (v/v) ethanol, anhydrous alcohol successively, finally be immersed into 5 ~ 6min in dimethylbenzene, air-dry take out microslide from dimethylbenzene after, when microslide is half-dried, drip neutral gum mounting liquid, mounting is preserved, and is then placed in basis of microscopic observation.
Wherein, the preparation method of the microslide of surface tiling breast cancer cell is as follows:
1) breast carcinoma cell strain of trypsinization in vitro culture, PBS solution washing and mixing.
2) hydro-extractor is opened, PBS containing breast carcinoma cell strain is added sample aperture, make total cell concentration of each sample aperture at 0.2 ~ 0.5M, preferably, the total cell concentration controlling each sample aperture is 0.3M, now, cell can be evenly distributed on microslide, medium density, is convenient to Microscopic observation, there will be cell overlap phenomenon time too much.PBS solution is abandoned in centrifugal rear suction, obtains the microslide of surface tiling breast cancer cell;
3) by after air-dry for above-mentioned microslide, acetone fixes 8 ~ 10min;
4) around tissue, use SABC stroke circle, astragal is from organizing about 3mm;
5) PBS washs 3 ~ 4min, then uses 3 ~ 5wt%H
2o
2soaking at room temperature 8 ~ 10min, finally with PBS washing 3 ~ 4 times, each 3 ~ 4min and get final product.
Wherein, in step (2), the centrifugal condition of Hettich is 2500 ~ 2800rcf, 6 ~ 8min.
Wherein, in step (4), the brown nitrite ion of described DAB is prepared as follows: (Fujian steps new to add DAB chromogenic reagent box in 850ul deionized water successively, article No. DAB-0031) in A liquid (DAB damping fluid) and B liquid (DAB substrate) and C liquid (DAB chromogen) each 50ul, after mixing, lucifuge is for subsequent use.
In step (5), 1% described acidic alcohol is prepared by the following method: add in the ethanol of 75% (v/v) in 36.5wt% ~ 38wt% concentrated hydrochloric acid, wherein, the ethanol of 75% (v/v) and the volume ratio of 36.5wt% ~ 38wt% concentrated hydrochloric acid are 100: 1.
When breast cancer cell for detecting in blood sample, step is similar, unlike, first extract the monocyte confluent monolayer cells in blood sample, then prepare the microslide of surperficial plated cells.The monocyte confluent monolayer cells extracted in blood sample adopts known method, as comprised the steps:
(A): collect peripheral blood sample with liquaemin anticoagulant tube, centrifugal, inhale and abandon upper plasma, obtain haemocyte;
(B): after haemocyte PBS steps A obtained dilutes mixing, add and placed in the centrifuge tube of lymphocyte separation medium in advance, the liquid got after centrifugal between cleer and peaceful lymphocyte separation medium is monocyte confluent monolayer cells, described monocyte confluent monolayer cells is added in PBS solution and washs, add PBS damping fluid after centrifugal and mix for subsequent use;
The method preparing surperficial plated cells is identical with the step of above-mentioned preparation table face tiling breast cancer cell.
Beneficial effect: the utility model provides a kind of kit for detecting breast cancer cell, structure is simple, easy to use, by with mouse anti human wide spectrum anti-cytokeratin Ab (AE1/AE3) for primary antibodie, with horseradish peroxidase-labeled rabbit anti-mouse antibody be two resist, by DAB and haematoxylin dyeing, make breast cancer cell in blood by specific stain.Method of the present utility model is simple to operate, and accuracy is good, has good application prospect.
Accompanying drawing explanation
Fig. 1 is the Color figure of breast cancer cell line mcf-7.
Fig. 2 is the peripheral blood Color figure (2A is the peripheral blood Color figure containing MCF-7,2B is the peripheral blood Color figure not containing MCF-7) containing breast cancer cell line mcf-7.
Fig. 3 is the marrow blood Color figure (3A is the marrow blood Color figure containing MCF-7,3B is the marrow blood Color figure not containing MCF-7) containing breast cancer cell line mcf-7.
Fig. 4 is the schematic diagram of kit of the present utility model.
Embodiment
The utility model provides a kind of kit for detecting breast cancer cell, as shown in Figure 4, comprise box body 1, lid 2, microslide, the primary antibodie reagent bottle 3 of mouse anti human wide spectrum anti-cytokeratin Ab is housed, two anti-reagent bottles 4 of horseradish peroxidase-labeled rabbit anti-mouse antibody are housed, DAB chromogenic reagent group, DAB nitrite ion storage bottle 5, be equipped with and haematoxylinly redye liquid reagent bottle 6, the color separation liquid reagent bottle 7 of 1% acidic alcohol is housed, the mounting liquid reagent bottle 8 of neutral gum is housed, wherein, box body comprises two-layer up and down, wherein, lower floor is separated into two cavitys, being respectively arranged with in two cavitys can the drawer of pull, each drawer outer wall is provided with draw ring 9, wherein, in a microslide cavity disposed therein, described DAB chromogenic reagent group is arranged in another cavity, DAB chromogenic reagent group comprises the reagent bottle that DAB damping fluid is housed, the reagent bottle that DAB substrate is housed and the reagent bottle of DAB chromogen is housed.The upper strata of box body is separated into six subregions, is respectively used to place primary antibodie reagent bottle 3, two anti-reagent bottle 4, DAB nitrite ion storage bottle 5, redye liquid reagent bottle 6, color separation liquid reagent bottle 7 and mounting liquid reagent bottle 8.Because microslide and DAB chromogenic reagent group frequency of utilization in once testing is not high, by microslide and DAB chromogenic reagent group being arranged at the lower floor of kit, the pollution caused when taking other reagent them can be avoided.
Sample and reagent source:
Breast carcinoma cell strain is MCF-7, purchased from Shanghai Chinese Academy of Sciences cell bank, primary antibodie is instant abcam mouse anti human wide spectrum anti-cytokeratin Ab (mouse anti human Anti-panCytokeratinantibody [AE1/AE3], article No. ab80826), two resist for horseradish peroxidase-labeled rabbit against murine two anti-(Fujian steps new, article No. 878973).
The preparation of reagent:
(1) the PBS pulvis of the preparation of PBS damping fluid: 0.01M, uses after adding 2000ml deionized water dissolving, pH7.2-7.4.
(2) preparation of DAB nitrite ion: DAB chromogenic reagent box is bought and stepped newly from Fujian, article No. DAB-0031, in 850ul deionized water, add the DAB damping fluid (A liquid) in DAB chromogenic reagent box, DAB substrate (B liquid) and DAB chromogen (C liquid) each 50ul during configuration successively, after mixing, lucifuge is for subsequent use.
The preparation of (3) 1% acidic alcohols: add in the ethanol of 75% (v/v) in 36.5wt% ~ 38wt% concentrated hydrochloric acid, wherein, the ethanol of 75% (v/v) and the volume ratio of 36.5wt% ~ 38wt% concentrated hydrochloric acid are 100: 1.
The utility model is described in detail below by specific embodiment.
The dyeing of embodiment 1 breast carcinoma cell strain.
(1) microslide of surface tiling breast cancer cell MCF-7 is first prepared: add 1mlPBS solution after being digested by the breast cancer cell line mcf-7 of in vitro culture, dilution mixes.
(2) Hettich hydro-extractor is opened, with the PBS application of sample containing breast cancer cell line mcf-7 in step (1), make total cell concentration of each sample aperture at 0.3M, PBS solution is abandoned in centrifugal rear suction, obtain the microslide of surface tiling breast cancer cell MCF-7, after air-dry, fix 10min with acetone.
(3) with SABC stroke circle around the tissue of the microslide obtained in step (2), astragal, from organizing about 3mm, washs 3min with PBS, then uses 3wt%H
2o
2soaking at room temperature 10min, then PBS washs 3 times, each 3min, obtains the microslide of surface tiling breast cancer cell.
(4) use instant abcam mouse anti human Anti-panCytokeratinantibody [AE1/AE3] (article No. ab80826) monoclonal antibody 1 ~ 1.5 as primary antibodie, microslide wet box in 37 degree of incubators of the breast cancer cell that tiled on the surface that itself and step (3) obtain hatches 1 hour; PBS washs 3 times, each 3min.
(5) drip horseradish peroxidase-labeled rabbit against murine two anti-1 ~ 1.5 to microslide, normal temperature wets in box and hatches 25min, and PBS washs 3 times, each 3min.
(6) microslide obtained to step (5) drips the brown nitrite ion of DAB 1.5 ~ 2 of fresh configuration, room temperature leaves standstill 5min, Microscopic observation, under low power lens (10 ×), visual field inner cell soaks cessation reaction with tap water after presenting brown colouring.
(7) the microslide haematoxylin that step (6) obtains is redyed 0.5min, after tap water is limpid to water, with 1% acidic alcohol color separation 1s; 1min is washed in water logging; Be immersed in 85% (v/v), 95% (v/v) ethanol, each 3min of anhydrous alcohol, the transparent 5min of dimethylbenzene successively, after taking out slice, thin piece, when slice, thin piece is half-dried, drip a neutral gum mounting liquid from dimethylbenzene, mounting is preserved.Basis of microscopic observation, as shown in Figure 1, as can be seen from the figure, breast cancer cell MCF-7 is colored as brown result.
Breast cancer cell line mcf-7 is added the staining examine carried out in normal circumference blood sample by embodiment 2.
1) collect peripheral blood sample 5ml with liquaemin anticoagulant tube, a small amount of PBS solution containing breast cancer cell line mcf-7 is added wherein, mixing.
2) centrifuge (3500 revs/min, 5 minutes), inhales and abandons upper plasma, about obtain 2 ~ 3ml haemocyte.
3) after remaining blood cell composition being mixed with 3mlPBS dilution, add and placed 15mlFicoll (lymphocyte separation medium in advance, Lymphoprep, Axis-ShieldPoCAS, Oslo, Norway) centrifuge tube in, the centrifugal 20min of horizontal centrifuge (22 DEG C, 2000 revs/min, 0 speed starts, and 0 speed reduction is to stopping).
4) from hydro-extractor, take out sample, extract monocyte confluent monolayer cells, and add in PBS solution after mixing, centrifugal (2000 revs/min, 10min), inhale and abandon original PBS, and add the new PBS dilution mixing of 2ml, for subsequent use.
5) by the monocyte confluent monolayer cells of blood sample that obtains according to the disposal methods of embodiment 1, dye.
After embodiment 2 detects, observations as shown in Figure 2.In Fig. 2 A, volume comparatively large and be brown colouring be breast cancer cell line mcf-7, small volume and what be not colored is the peripheral blood cells of people.Be the testing result of the peripheral blood sample not adding breast cancer cell line mcf-7 in Fig. 2 B, join as the moon.Therefore, as can be seen from result, colouring method of the present utility model successfully can carry out differential dyeing to the breast cancer cell added in normal plasma cell, contributes to the micrometastasis differentiating whether to have breast cancer cell in peripheral blood, thus significant to the Index for diagnosis of breast cancer.
Breast carcinoma cell strain is added the staining examine carried out in marrow blood sample by embodiment 3.
1) collect marrow blood sample 5ml with liquaemin anticoagulant tube, a small amount of PBS solution containing breast cancer cell line mcf-7 is added wherein, mixing.
2) centrifuge (3500 revs/min, 5 minutes), inhales and abandons upper plasma, about obtain 2 ~ 3ml haemocyte.
3) after remaining blood cell composition being mixed with 3mlPBS dilution, add and placed 15mlFicoll (lymphocyte separation medium in advance, Lymphoprep, Axis-ShieldPoCAS, Oslo, Norway) centrifuge tube in, the centrifugal 20min of horizontal centrifuge (22 DEG C, 2000 revs/min, 0 speed starts, and 0 speed reduction is to stopping).
4) from hydro-extractor, take out sample, extract monocyte confluent monolayer cells, and add in PBS solution after mixing, centrifugal (2000 revs/min, 10min), inhale and abandon original PBS, add the PBS dilution mixing that 2ml is new, for subsequent use.
5) by the monocyte confluent monolayer cells of marrow blood sample that obtains according to the disposal methods of embodiment 1, dye.
After embodiment 3 detects, observations as shown in Figure 3.In Fig. 3 A, volume comparatively large and be brown colouring be breast cancer cell line mcf-7, small volume and what be not colored is the Blood cells in bone marrow of people.Be the testing result of the marrow blood sample not adding breast carcinoma cell strain in Fig. 3 B, join as the moon.Therefore, colouring method of the present utility model successfully can carry out differential dyeing to the breast cancer cell added in marrow blood, contributes to the micrometastasis differentiating whether to have breast cancer cell in marrow blood, thus significant to the Index for diagnosis of breast cancer.
Claims (3)
1. one kind for detecting the staining kit of breast cancer cell, it is characterized in that, comprise box body, lid, microslide, the primary antibodie reagent bottle of mouse anti human wide spectrum anti-cytokeratin Ab is housed, two anti-reagent bottles of horseradish peroxidase-labeled rabbit anti-mouse antibody are housed, DAB chromogenic reagent group, DAB nitrite ion storage bottle, be equipped with and haematoxylinly redye liquid reagent bottle, the color separation liquid reagent bottle of 1% acidic alcohol is housed, the mounting liquid reagent bottle of neutral gum is housed, wherein, described box body comprises two-layer up and down, wherein, lower floor is separated into two cavitys, being respectively arranged with in two cavitys can the drawer of pull, wherein, in a described microslide cavity disposed therein, described DAB chromogenic reagent group is arranged in another cavity, described DAB chromogenic reagent group comprises the reagent bottle that DAB damping fluid is housed, the reagent bottle that DAB substrate is housed and the reagent bottle of DAB chromogen is housed, the upper strata of described box body is separated into six subregions, is respectively used to place primary antibodie reagent bottle, two anti-reagent bottles, DAB nitrite ion storage bottle, redye liquid reagent bottle, color separation liquid reagent bottle and mounting liquid reagent bottle.
2. staining kit according to claim 1, is characterized in that, described DAB nitrite ion storage bottle is brown storage bottle.
3. staining kit according to claim 1, is characterized in that, described the drawer outside of pull can be provided with draw ring.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201520684125.0U CN205049576U (en) | 2015-09-06 | 2015-09-06 | A dyeing kit for detecting breast cancer cell |
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| CN201520684125.0U CN205049576U (en) | 2015-09-06 | 2015-09-06 | A dyeing kit for detecting breast cancer cell |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105223058A (en) * | 2015-09-06 | 2016-01-06 | 江苏省人民医院 | A kind of colouring method of breast cancer cell and application thereof and staining kit |
| CN105785006A (en) * | 2016-04-21 | 2016-07-20 | 合肥市第二人民医院 | Method for warning breast cancer metastasis based on BCL-6 and ZEB-1 as markers |
-
2015
- 2015-09-06 CN CN201520684125.0U patent/CN205049576U/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105223058A (en) * | 2015-09-06 | 2016-01-06 | 江苏省人民医院 | A kind of colouring method of breast cancer cell and application thereof and staining kit |
| CN105785006A (en) * | 2016-04-21 | 2016-07-20 | 合肥市第二人民医院 | Method for warning breast cancer metastasis based on BCL-6 and ZEB-1 as markers |
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Granted publication date: 20160224 Termination date: 20170906 |
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