CN105510600A - Method for detecting ER gene of peripheral blood circulating tumor cells of patient suffering from advanced breast cancer - Google Patents
Method for detecting ER gene of peripheral blood circulating tumor cells of patient suffering from advanced breast cancer Download PDFInfo
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Abstract
The invention discloses a method for detecting an ER gene of peripheral blood circulating tumor cells of a patient suffering from advanced breast cancer. The method comprises the steps of conducting separation with a membrane filtration device, so that CTC in peripheral blood of the patient suffering from advanced breast cancer is obtained; identifying the peripheral blood circulating tumor cells of the patient suffering from advanced breast cancer through the cellular immunofluorescence technique; preparing thin layer sections through the cell paraffin block technique; further detecting the ER expression condition of the peripheral blood circulating tumor cells of the patient suffering from advanced breast cancer through the immunohistochemical technique. According to the method, CTCs are separated and enriched by means of the ISET technique, and the CTCs are identified through the cellular immunofluorescence technique, so that the difficulty existing in CTCs identification conducted through the pure ISET technique and false negativeness existing in CTCs identification conducted through the pure immunological detection technique are overcome. The technique is not high in equipment requirement, the method is easy to grasp, and real-time monitoring can be conducted. By means of the technical method, the ER expression condition of the patient suffering from advanced breast cancer can be detected without picking breast cancer tissues.
Description
Technical field
The present invention relates to a kind of new method detecting ER gene, detect the method for advanced breast cancer patient ER gene especially by peripheral blood.
Background technology
The estrogen receptor (Estrogenreceptor, ER) included in treatment guidelines as prognostic factor at present has become most important index when formulating patient with breast cancer's endocrine therapy scheme.The detection of existing expressions of ER needs the tumor tissues sample being obtained primary tumor or metastatic tumor by modes such as operation or aspiration biopsies, right instead of each patient can obtain tumor tissues sample, and continuous print detection can not be carried out over the course for the treatment of, limiting it becomes effective curative effect monitoring index.But the circulating tumor cell come off into blood can provide potential real-time tumor specimen to us, its mutation status may be used for guiding treatment equally.
Circulating tumor cell (Circulatingtumorcell, CTC) be come off from entity tumor and enter the tumour cell of peripheral blood circulation, it is present in the Several Kinds of Malignancies such as breast cancer, prostate cancer, colorectal cancer, its detect for assessment tumor patient especially late tumor patient prognosis and select suitable individualized treatment to have important clinical meaning.Because CTC detects, there is Wicresoft, the feature such as real-time, be called as " liquid biopsy ".
The main method that current CTCs detects comprises the immunology detection technology of dependence tumor related marker quality testing survey and the membrane filtration technique (Isolatingbysizeofepithelialtumorcells, ISET) based on morphology enrichment.The former is based on the different antigen marker of cell surface expression, and specific antibody EpCAM, CK etc. for TCSA are coated in magnetic bead surfaces to complete the specific recognition of tumour cell, by externally-applied magnetic field screening enrichment CTCs.The advantage of this technology is that specificity is higher, existing commercial semi-automatic checkout equipment (CellSearch); But the disappearance that tumour specific antigen is expressed can cause false negative result.The latter is the difference according to cell size, utilizes the method for filtering by tumour cell larger for volume screening enrichment.The advantage of this technology is that method is simple, cheap, and the CTCs being separated acquisition has activity and can be used for follow-up study; But owing to lacking Morphologic Diagnosis goldstandard, this beneficiation technologies specificity is relatively poor.
Summary of the invention
Be not suitable for tissue specimen draw materials to overcome conventional recycle tumour cell detection technique and advanced breast cancer patient--and then cannot the deficiency of assess patient ER state, patent of the present invention provides a kind of detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene: utilize film filter to be separated the CTC obtained in advanced breast cancer peripheral blood in patients; Utilize cellular immunofluorescence technical appraisement advanced breast cancer peripheral blood in patients circulating tumor cell; Use the section of cell block fabrication techniques thin layer; And then the ER expression of advanced breast cancer peripheral blood in patients circulating tumor cell is detected by immunohistochemistry technology.
The method concrete steps are as follows:
One, utilize film filter to be separated and obtain advanced breast cancer peripheral blood in patients circulating tumor cell:
1, gather advanced breast cancer disease peripheral blood in patients: median basilic vein 10ml, be divided into after 2 pipes write identical numbering, called after tests 1 group and experiment 2 groups respectively;
2,2 pipe peripheral blood sample are carried out pre-service respectively: by the peripheral blood sample 10 times dilution gathered, diluent ingredient: 1mmol/lEDTA+0.1%BSA, after dilution, fix peripheral blood sample 10 minutes with 4% paraformaldehyde;
3, utilize film filter to be separated peripheral blood sample, enrichment Peripheral Circulation tumour cell: pretreated peripheral blood sample joined in the blood sample container of film filter, makes it rely on gravity natural filtration;
4, after filtration terminates, from film filter, take off filter, PBS rinses 2-3 time, and Peripheral Circulation tumour cell is trapped within filter membrane.
Two, cellular immunofluorescence technical appraisement advanced breast cancer peripheral blood in patients circulating tumor cell is utilized:
1, be separated to experiment 1 group in the filter of acquisition Peripheral Circulation tumour cell and add Cytoperm/Cytofix reagent 300 μ l, fixing, perforation 15min;
2, PBS rinsing 3 times, each 5 minutes, adds washbuffer300 μ l afterwards, closes 30min;
3, add CK8/18/19, CD45 primary antibodie suspension 300 μ l, two kinds of primary antibodies are all with washbuffer dilution, and the final concentration after mixing is 1:250, incubated at room temperature 45min;
4, Washbuffer washs 3 times, each 5 minutes, adds two anti-suspension 300 μ l afterwards, and two kind two anti-all with washbuffer dilution, and the final concentration after mixing is 1:500, and under room temperature, lucifuge hatches 30min;
5, Washbuffer washs 3 times, each 5 minutes, adds Hoechst300 μ l afterwards, washes transfect cell core 5min;
6, PBS rinsing 2 times, each 3 minutes, takes out filter membrane afterwards and is placed on microslide, drip 10 μ l anti-fluorescent quenching confining liquid mounting;
7, observations in 2h under fluorescent microscope, takes pictures, records CTC situation, determine whether there is CTC; Then proceed next step detection as observed CTC, otherwise stop detecting.
Three, cell block fabrication techniques CTC cell block is used:
1, from filtration unit, take off the filter of the identical blood sample numbering of experiment 2 groups, to open and to remove filter suitable for reading, circulating tumor cell dyeing liquor is joined in filter, dyeing 2min, PBS wash buffer is clean, takes off filter membrane, be placed on microslide with ophthalmology tweezers, examine under a microscope after suitable drying, alleged occurrence CTC;
2, CTC cell block is made;
A, decolouring: taken off by the filter membrane on above-mentioned microslide, be placed in destainer and soak 4-6 hour, sloughs CTC dyeing liquor, and described destainer is: 95% alcohol and 100% dimethylbenzene by volume 1:1 mix;
B, parcel: soak and terminate rear taking-up filter membrane, wrap up with thieving paper;
C, fixing: wrappage to be placed in 10% neutral formalin, fixing 4-6h.
D, waxdip embed: take out wrappage after fixing end, dehydration is placed in paraffin embedding box, and waxdip embedding makes cell paraffin mass.
E, thin layer are cut into slices: with microtome by cell paraffin mass serial section, make the thin layer section that thickness is 2-4 μm.
Four, the ER expression of advanced breast cancer peripheral blood in patients circulating tumor cell is detected by immunohistochemistry technology:
1. roasting sheet: the section of CTC wax stone thin layer bakes sheet 2 hours in 60 DEG C of constant temperature ovens;
2. dewax: cut into slices and place 5 minutes respectively in two bottles of dimethylbenzene;
3. aquation: section is placed respectively 1 minute (slightly adjusting depending on indoor temperature situation) successively in absolute ethyl alcohol, 95% ethanol, 85% ethanol: tap water, distilled water flushing;
4. antigen retrieval: the reparation liquid EDTA in pressure cooker is boiled, put into section and cover high-pressure pot cover and pressure valve to the reparation liquid that submerges completely, be heated to pressure cooker jet latter 1.5 minutes, from fire, pressure cooker is slightly cooled and opens pot cover, cooling naturally in reparation liquid of cutting into slices;
5. distilled water flushing;
6. remove endogenous peroxydase: with 3%H2O2 effect section 10 minutes;
7. distilled water flushing;
8.PBS embathe 5 times each 1.5 minutes;
9. drip ER primary antibodie, 4 DEG C of refrigerator overnight, then put it into PBS and embathe 5 times, embathe 1.5 minutes at every turn;
10. drip two to resist;
20 minutes are hatched in 11.37 DEG C of water-baths;
12.PBS embathe 5 times each 1.5 minutes;
Control under 13. microscopes, DAB develops the color;
14. tap water, haematine dye liquor redyes 2 minutes, and hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant;
15. tap water 5 minutes;
16.75%-95%-100% gradient alcohol dehydration, dimethylbenzene is transparent, neutral Instant cement mounting;
17. cell pathology expert diagosis, interpretation ER expression.
Utilize the film filter described in cellular immunofluorescence technical appraisement Peripheral Circulation tumour cell step: comprise filter, blood sample container, waste liquid cylinder and iron stand, described iron stand is provided with base, stand and support, described blood sample container by Bracket setting in iron stand top, the below of blood sample container is filter, filter is by transfusion device UNICOM to waste liquid cylinder, and waste liquid cylinder is arranged on base.
Described filter comprises that filter is suitable for reading, filter membrane, carry filter membrane platform and filter end opening, and filter membrane is placed in and carries on filter membrane platform; Filter is suitable for reading connects blood sample container, and filter end opening connects waste liquid cylinder by transfusion device.
Described filter membrane is that hydrophobic material is made, and it is evenly covered with the filter opening that bore is 10 microns.
Beneficial effect: by means of ISET technology separation enrichment CTCs, rely on cellular immunofluorescence technical appraisement CTCs, the difficulty that simple ISET technical appraisement CTCs exists can not only be overcome, also can catch epithelium antigen and express the tumour cell weakening or lack, namely overcome the false negative that simple immunology detection technology exists.Meanwhile, this technical equipment is less demanding, and method is easy to grasp, and energy Real-Time Monitoring, having clinical application may.By this technical method, the breast cancer tissue that need not draw materials can detect advanced breast cancer patient ER expression.This technology belongs to Wicresoft even without wound, and can detect in real time.
Accompanying drawing explanation
Fig. 1 is film filter structural representation of the present invention;
Fig. 2 is the structural representation cut-open view of the filter of film filter of the present invention;
Fig. 3 is the structural representation of the filter filter membrane of film filter of the present invention;
Fig. 4 is that advanced breast cancer cancer peripheral blood in patients is separated the circulating tumor cell striograph obtained;
Fig. 5 is advanced breast cancer peripheral blood in patients circulating tumor cell SABC ER colored graph picture.
1. iron stand 2. blood sample container 3. filter 4. transfusion device 5. waste liquid cylinder 6. filter, 7. filter membrane suitable for reading, 8. years filter membrane platforms, 9. filter end opening, 10. filter opening, 11. bases, 12. stands, 13 supports in figure.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
Use this technical method to be separated and obtain and identify 8 routine advanced breast cancer cancer patients (detect 8 routine normal person's samples simultaneously and do negative control) Peripheral Circulation tumour cell, detect its ER expression.
One, film filter is utilized to be separated the CTC obtaining and cannot obtain in the advanced breast cancer peripheral blood in patients of tissue specimen:
Gather advanced breast cancer peripheral blood in patients 10ml, be divided into 2 pipes, same blood sample of patient writes identical numbering, and called after tests 1 group and experiment 2 groups respectively; Then 2 pipe peripheral blood sample are carried out pre-service respectively: by the peripheral blood sample 10 times dilution gathered, diluent ingredient: 1mmol/lEDTA+0.1%BSA), fix peripheral blood sample 10 minutes with 4% paraformaldehyde after dilution; In fixing interval, assembling film isolated by filtration tumour cell (ISET) easy device (as shown in Figure 1, 2, 3): this filtration unit is made up of filter 3, filter membrane 7, blood sample container 2, waste liquid cylinder 5, iron stand 1, film filter is utilized to be separated peripheral blood sample, enrichment Peripheral Circulation tumour cell: soak filter 3 with 10mlPBS, then pretreated peripheral blood sample is joined in the blood sample container 2 of film filter, make it rely on gravity natural filtration; After filtration terminates, from film filter, take off filter 3, PBS rinse 2-3 time, peripheral blood CTC is trapped within filter membrane 7.
Tumor cell diameter is generally greater than 15 microns, and haemocyte (comprising red blood cell, leucocyte) diameter is generally less than 10 microns, therefore when the peripheral blood containing CTC after filtering, haemocyte can be filtered across because diameter is less than filter opening 10, and CTC is trapped within filter membrane 7 because diameter is greater than filter opening 10.
Two, cellular immunofluorescence technical appraisement advanced breast cancer peripheral blood in patients circulating tumor cell is utilized:
Obtain (testing 1 group) in the filter 3 of Peripheral Circulation tumour cell to separation and add Cytoperm/Cytofix reagent 300 μ l, fixing, perforation 15min; PBS rinsing 3 times, each 5 minutes, adds washbuffer300 μ l afterwards, closes 30min; Add CK8/18/19, CD45 primary antibodie suspension 300 μ l, two kinds of primary antibodies are all with washbuffer dilution, and the final concentration after mixing is 1:250, incubated at room temperature 45min; Washbuffer washs 3 times, each 5 minutes, adds two anti-suspension 300 μ l afterwards, and two kind two anti-all with washbuffer dilution, and the final concentration after mixing is 1:500, and under room temperature, lucifuge hatches 30min; Washbuffer washs 3 times, each 5 minutes, adds Hoechst300 μ l afterwards, washes transfect cell core 5min; PBS rinsing 2 times, each 3 minutes, takes out filter membrane 7 afterwards and is placed on microslide, drip 10 μ l anti-fluorescent quenching confining liquid mounting; Observations in 2h under fluorescent microscope, take pictures, record CTC situation (as experimental group 1 observe CTC then application experiment group 2 carry out next step detect).
Testing result: 8 routine healthy volunteers all do not find circulating tumor cell; In 8 routine advanced breast cancer peripheral blood in patients, there are 6 examples to detect that CTC exists.
Three, the section of cell block fabrication techniques thin layer is used:
Take off from filtration unit in experiment 2 groups and the filter 3 that CTC numbers detected, open and remove filter suitable for reading 6, circulating tumor cell dyeing liquor is joined in filter 3, dyeing 2min, PBS wash buffer is clean, takes off filter membrane 7, be placed on microslide with ophthalmology tweezers, examine under a microscope after suitable drying, alleged occurrence CTC;
Filter membrane 7 on above-mentioned microslide is taken off, be placed in destainer soak 4 ?6 hours, slough CTC dyeing liquor, described destainer is: 95% alcohol and 100% dimethylbenzene by volume 1:1 mix; Immersion terminates rear taking-up filter membrane 7, wraps up with thieving paper; Wrappage is placed in 10% neutral formalin, fixing 4-6h; Take out wrappage after fixing end, dehydration is placed in paraffin embedding box, and waxdip embedding makes cell paraffin mass; With microtome by cell paraffin mass serial section, make the thin layer section that thickness is 2-4 μm.
Four, the ER expression of advanced breast cancer peripheral blood in patients circulating tumor cell is detected by immunohistochemistry technology:
The paraffin section applying row immunohistochemical method obtained is detected the expression of ER gene, to cut into slices in 60 DEG C of constant temperature ovens roasting sheet 2 hours, cut into slices and place 5 minutes respectively in two bottles of dimethylbenzene, section is placed respectively 1 minute (slightly adjusting depending on indoor temperature situation) successively in absolute ethyl alcohol, 95% ethanol, 85% ethanol: tap water, distilled water flushing;
Antigen retrieval: according to first antibody instructions and specific experiment operating experience, diverse ways is used to carry out antigen retrieval to experimental slides, this experiment uses the reparation of EDTA (PH=9.0) hot high pressure: boiled by the reparation liquid (EDTA) in pressure cooker on electromagnetic oven, put into section and cover high-pressure pot cover and pressure valve to the reparation liquid that submerges completely, 800W is heated to pressure cooker jet latter 1.5 minutes, from fire, pressure cooker is slightly cooled and opens pot cover, cut into slices and repairing cooling naturally in liquid, then distilled water flushing is used, endogenous peroxydase is removed: (adopt the detection system of peroxidase after flushing, endogenous peroxydase Seal treatment must be carried out, if do not processed, red blood cell in tissue, granulocyte can disturb the judgement of coloration result) this experiment 3%H2O2 effect section 10 minutes, coloration result collection antigen is preserved and does not all affect, sealing effect is more remarkable, distilled water flushing, PBS embathe 5 times each 1.5 minutes, drip ER primary antibodie, 4 DEG C of refrigerator overnight, then put it into PBS and embathe 5 times, embathe 1.5 minutes at every turn, drip two resist, hatch 20 minutes in 37 DEG C of water-baths, PBS embathe 5 times each 1.5 minutes, control under microscope, DAB develops the color, tap water, haematine dye liquor redyes 2 minutes, and hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant, tap water 5 minutes, 75%-95%-100% gradient alcohol dehydration, dimethylbenzene is transparent, neutral Instant cement mounting, 2-3 name cell pathology expert diagosis, interpretation ER expression.
Testing result: 8 routine healthy volunteers all do not find circulating tumor cell; At the CTC that 8 routine advanced breast cancer peripheral blood in patients detect, wherein through SABC, 6 examples detect that it has ER to express, and positive rate is 75.0% (table 1).
Fig. 4 is that Peripheral Blood In Patients With Breast Cancer is separated the circulating tumor cell striograph obtained, and its nucleus is comparatively large, and nuclear shapes is irregular; High nucleocytoplasmic ratio.
Fig. 5 is Peripheral Blood In Patients With Breast Cancer circulating tumor cell ER immunohistochemistry, cell membrane and cytoplasm xanthochromia.Judge positive degree according to its dye distribution and intensity, cell color accounts for cell and is less than 10% for (-), and painted 10%-25% be (+), and painted 25%-50% is (++), and painted to be greater than 50% be (+++).
Table 1 embodiment testing result (ER)
Claims (8)
1. a detection method for advanced breast cancer peripheral blood in patients circulating tumor cell ER gene, is characterized in that, utilizes film filter to be separated the CTC obtained in advanced breast cancer peripheral blood in patients; Qualification advanced breast cancer peripheral blood in patients circulating tumor cell; Use the section of cell block fabrication techniques thin layer; And then detect the ER expression of advanced breast cancer peripheral blood in patients circulating tumor cell.
2. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 1, it is characterized in that, described film filter comprises filter (3), blood sample container (2), waste liquid cylinder (5) and iron stand (1), described iron stand (1) is provided with base (11), stand (12) and support (13), described blood sample container (2) is arranged at iron stand (1) top by support (13), the below of blood sample container (2) is filter (3), filter (3) by transfusion device (4) UNICOM to waste liquid cylinder (5), waste liquid cylinder (5) is arranged on base (11).
3. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 2, it is characterized in that: described filter (3) comprises filter (6) suitable for reading, filter membrane (7), carries filter membrane platform (8) and filter end opening (9), filter membrane (7) is placed in and carries on filter membrane platform 8; Filter (6) suitable for reading connects blood sample container (2), and filter end opening (9) connects waste liquid cylinder (5) by transfusion device (4).
4. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 3, it is characterized in that: described filter membrane (7) is made for hydrophobic material, it is evenly covered with the filter opening (10) that bore is 10 microns.
5. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 1, is characterized in that, the described film filter that utilizes is separated acquisition advanced breast cancer peripheral blood in patients circulating tumor cell, and step is as follows:
1) advanced breast cancer disease peripheral blood in patients is gathered: median basilic vein 5ml;
2) peripheral blood sample is carried out pre-service: by the peripheral blood sample 10 times dilution gathered, diluent ingredient: 1mmol/lEDTA+0.1%BSA, after dilution, fix peripheral blood sample 10 minutes with 4% paraformaldehyde;
3) utilize film filter to be separated peripheral blood sample, enrichment Peripheral Circulation tumour cell: pretreated peripheral blood sample joined in the blood sample container (2) of film filter, makes it rely on gravity natural filtration;
4) after filtration terminates, take off filter (3) from film filter, PBS rinses 2-3 time, and Peripheral Circulation tumour cell is trapped within filter membrane (7).
6. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 1, is characterized in that, described qualification advanced breast cancer peripheral blood in patients circulating tumor cell, and step is as follows:
1) obtain to separation in the filter (3) of Peripheral Circulation tumour cell and add Cytoperm/Cytofix reagent 300 μ l, fixing, perforation 15min;
2) PBS rinsing 3 times, each 5 minutes, adds washbuffer300 μ l afterwards, closes 30min;
3) add CK8/18/19, CD45 primary antibodie suspension 300 μ l, three kinds of primary antibodies are all with washbuffer dilution, and the final concentration after mixing is 1:250, incubated at room temperature 45min;
4) Washbuffer washs 3 times, each 5 minutes, adds two anti-suspension 300 μ l afterwards, and three kind two anti-all with washbuffer dilution, and the final concentration after mixing is 1:500, and under room temperature, lucifuge hatches 30min;
5) Washbuffer washs 3 times, each 5 minutes, adds Hoechst300 μ l afterwards, washes transfect cell core 5min;
6) PBS rinsing 2 times, each 3 minutes, takes out filter membrane (7) afterwards and is placed on microslide, drips 10 μ l anti-fluorescent quenching confining liquid mounting;
7) observations in 2h under fluorescent microscope, takes pictures, records CTC situation, determine whether there is CTC.
7. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 1, is characterized in that, described utilization cell block fabrication techniques thin layer section, and step is as follows:
1) after peripheral blood filters, filter (3) is taken off from filtration unit, open and remove filter (6) suitable for reading, joined by circulating tumor cell dyeing liquor in filter (3), dyeing 2min, PBS wash buffer is clean, filter membrane (7) is taken off with ophthalmology tweezers, be placed on microslide, examine under a microscope after suitable drying, alleged occurrence CTC;
2) thin layer section is made:
A, decolouring: taken off from microslide by the above-mentioned filter membrane with CTC (7), be placed in 95% alcohol and the 100% dimethylbenzene destainer that 1:1 mixes by volume soaks 4-6 hour, sloughs CTC dyeing liquor;
B, parcel: soak and terminate rear taking-up filter membrane (7), wrap up with thieving paper;
C, fixing: wrappage to be placed in 10% neutral formalin, fixing 4-6h;
D, waxdip embed: take out wrappage after fixing end, dehydration is placed in paraffin embedding box, and waxdip embedding makes cell paraffin mass;
E, thin layer are cut into slices: with microtome by cell paraffin mass serial section, make the thin layer section that thickness is 2-4 μm.
8. the detection method of advanced breast cancer peripheral blood in patients circulating tumor cell ER gene according to claim 1, is characterized in that, the ER expression of described detection advanced breast cancer peripheral blood in patients circulating tumor cell, and step is as follows:
1) roasting sheet: the section of CTC wax stone thin layer bakes sheet 2 hours in 60 DEG C of constant temperature ovens;
2) dewax: cut into slices and place 5 minutes respectively in two bottles of dimethylbenzene;
3) aquation: section is placed 1 minute respectively successively in absolute ethyl alcohol, 95% ethanol, 85% ethanol, tap water, distilled water flushing;
4) antigen retrieval: the reparation liquid EDTA in pressure cooker is boiled, put into section and cover high-pressure pot cover and pressure valve to the reparation liquid that submerges completely, be heated to pressure cooker jet latter 1.5 minutes, from fire, pressure cooker is slightly cooled and opens pot cover, cooling naturally in reparation liquid of cutting into slices;
5) distilled water flushing;
6) endogenous peroxydase is removed: with 3%H2O2 effect section 10 minutes;
7) distilled water flushing;
8) PBS embathe 5 times each 1.5 minutes;
9) drip ER primary antibodie, 4 DEG C of refrigerator overnight, then put it into PBS and embathe 5 times, embathe 1.5 minutes at every turn;
10) drip two to resist;
11) 20 minutes are hatched in 37 DEG C of water-baths;
12) PBS embathe 5 times each 1.5 minutes;
13) control under microscope, DAB develops the color;
14) tap water, haematine dye liquor redyes 2 minutes, and hydrochloride alcohol breaks up, and ammoniacal liquor returns indigo plant;
15) tap water 5 minutes;
16) 75%-95%-100% gradient alcohol dehydration, dimethylbenzene is transparent, neutral Instant cement mounting.
17) cell pathology expert diagosis, interpretation ER expression.
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