CN103105324A - Same-section multi-target protein immunohistochemical or immunofluorescent labeling method - Google Patents
Same-section multi-target protein immunohistochemical or immunofluorescent labeling method Download PDFInfo
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- CN103105324A CN103105324A CN201310031801XA CN201310031801A CN103105324A CN 103105324 A CN103105324 A CN 103105324A CN 201310031801X A CN201310031801X A CN 201310031801XA CN 201310031801 A CN201310031801 A CN 201310031801A CN 103105324 A CN103105324 A CN 103105324A
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Abstract
The invention provides a same-section multi-target protein immunohistochemical or immunofluorescent labeling method. The method comprises the following steps of: unfolding slices in corresponding liquid in the process of slicing a tissue wax block, selecting two continuously connected slices in the process of catching a target slice, placing the back surface of the first slice upwards in the process of catching the slice by employing a glass slide, and placing the front surface upwards in the process of catching the second slice, so that the condition that the surfaces of the two tissue slice for staining belong to the same slicing surface is ensured; and respectively performing immunohistochemical or immunofluorescent staining on different target proteins for two slices from the same slicing surface, performing overlap comparison on the tissue staining result in a same area, so as to realize existential analysis of a same cell or tissue site multi-target protein. The effect of performing multi-target protein labeling on the same cell or tissue site to be detected is achieved.
Description
Technical field
The invention belongs to field of pathology, particularly, the present invention relates to a kind of method of same many target point proteins of tangent plane SABC or immunofluorescence dyeing mark.
Background technology
Tumor markers be tumour cell cancerate, produce in the process such as Emergence and Development, infiltration, transfer with the closely-related physiological activator of growth of tumour cell metabolism.They often are detected in embryonic tissue, do not exist and seldom be present in normal adult tissue, but the content in adult's tumor tissues substantially exceeds the content in normal structure, by detecting its existence in corresponding cell or tissue and the variation of concentration thereof, determine not know the primary tumo(u)r of the metastatic tumour of originating, can carry out diagnosis, the classification of tumour and characteristic thereof, the monitoring of operation, chemotherapy, radiotherapy, give simultaneously the variation according to index before and after treatment, carry out prognosis, the result for the treatment of of assess patient.Tumor markers is made a definite diagnosis level to the raising clinical tumor and is had important reference value as the foundation of pathological diagnosis.Along with the reach of science, the research of tumor markers is more and more deep at present, and its physiological significance to tumour cell obtains announcement progressively, and simultaneously, the discovery of the new tumor markers of high specificity becomes an important direction in present tumor research.
Along with going deep into of research, the classification of tumor markers is more and more accurate, and the use in conjunction of multi-tumor Markers can improve efficient and the accuracy rate of detection greatly.At present the pathological diagnosis standard of tissue is mainly according to immunohistochemistry technique, by dye dyeing with a tumor markers of HE, carries out the diagnosis of tumour.Defective in view of the color classification of immunohistochemical staining also can't realize carrying out the discriminating of multi-tracer at a histotomy at present.If employing serial section, carry out the dye marker of tumor markers in the difference section, although can realize the identification of many tumor markers, but the defective of bringing due to self thickness of section, make the different labels dyeing in same site may be on different cells, the same intracellular unlike signal thing level of reaction that can't entirely accurate.
Tumor stem cell (cancer stem cells, CSC) is the cell that has self-renewal capacity in tumour and can produce heterogeneous tumour cell, is the major reason that tumour is difficult to cure.In recent years to CSC research deeply and the progress of stem cell isolation technics, make people to malignant tumour, new cognition arranged, and find that CSC plays an important role in generation, growth, infiltration, recurrence and the transfer of tumour.By separation and purification CSCs, can study evolution, transfer and the resistance to the action of a drug mechanism etc. of its biological characteristics such as heterogeneous tumour; Identify the genome of CSCs, seek the specificity target position of CSCs, development of new is for the medicine of CSCs.To its characteristic research deeply help illustrate that CSCs forms in tumour, growth, infiltrate, recurrence and shift in mechanism of action, and then seek and thoroughly kill the method for CSCs to reach the purpose of final healing tumour, for the clinical treatment of tumour represents a brand-new visual angle, for neoplasm targeted therapy finds novel targets, make the healing tumour become possibility.So far the researchist successively successfully isolates tumor stem cell from the malignant tumours such as breast cancer, brain tumor, prostate cancer, colon cancer, lung cancer, oophoroma, melanoma, cancer of pancreas, liver cancer.The successful separation of tumor stem cell makes the research of oncology stride into the CSCs research epoch.
The separation of tumor stem cell is identified and is mainly relied in the identification of the distinctive surface molecular label of CSC and be different from the biological characteristics of common tumour cell.The tumor stem cell surface molecular is marked with two classes: a kind of is the mark of normal stem cell/CFU-GM.As CD133, Nanog etc.Research is found to have the CD133+ cell in human hepatoma cell strain and liver in situ cancerous tissue.Another kind of CSC mark may be the mark relevant with development, the transfer of tumour, as EpCAM, CD90
+, CD45
+At present, realize the method for difference tumour cell, tumor stem cell by the method for the special acceptor of antibody-mediated identification cell surface, mainly contain flow cytometer cell instrument and immunofluorescence technique.
The technology of the many antigenic marks of present above-mentioned needs also is difficult to realize with immunohistochemistry technique, can carry out at most the mark of two kinds of antibody in present same section, namely and mark alkaline phosphatase generation bluish violet brown by the generation of mark horseradish enzyme labeling distinguished, but both on the differentiation of color and technical operation, certain difficulty is arranged, use simultaneously the application that two kinds of different enzymic-labelled antibodies carry out immunohistochemical staining few.And how immunohistochemistry technique to be hospital pathology department use proven technique realizes that many antigens, multiple goal albumen carry out immunohistochemical markers on the same tangent plane of section, and the development of pathological diagnosis is had important effect.The invention provides and a kind ofly can carry out method and the technology of many target spots immunohistochemical markers on the same tangent plane of section.
Summary of the invention
The object of the present invention is to provide a kind of method of same many target point proteins of tangent plane SABC or immunofluorescence dyeing mark, the inventive method has realized that many antigens, multiple goal albumen carry out the dye marker of SABC on the same tangent plane of section, and the development of pathological diagnosis is had important effect.
For achieving the above object, technical scheme provided by the invention is as follows:
A kind of method of same many target point proteins of tangent plane SABC or immunofluorescence dyeing mark, when paraffin embedded tissues is cut into slices, get two continuous sections, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that namely are used for dyeing belong to same tangent planes.
To belonging to two sections of same tangent plane, carry out respectively SABC or the immunofluorescence dyeing of different target point proteins, during observation, the tissue staining result of getting corresponding the same area overlaps comparison, realizes the existence analysis of the many target point proteins in same cell or tissue site.
When described two continuous sections refer to histotomy, be close to two sections together, the front of the back side of first section and second section is the two sides of the same tissue after single solution for diverse problems is opened.
When the front of described section refers to cut into slices, in the face of tangent plane operator's one side, the described back side is near the one side of tissue back to the operator.
Described target point protein refers to have in pathology detection the target protein of diagnosis effect.
Remarkable advantage of the present invention: a kind of method that the object of the present invention is to provide same many target point proteins of tangent plane SABC or immunofluorescence dyeing mark, the inventive method has realized that many antigens, multiple goal albumen carry out the dye marker of SABC on the same tangent plane of section, and the development of pathological diagnosis is had important effect.
Embodiment
The method of a kind of same many target point proteins of tangent plane immunohistochemical staining mark of the present invention comprises the steps:
The wax stone that will contain tumor tissues is fixed on histotome, tissue is cut into slices, when blade runs to destination organization, begin to choose, one serial section that is chained together is put into the ethanolic solution 1 minute to 5 minutes of 40%-60%, and then the hot water of putting into 40-60 ° of C is opened up sheet, choose two sections that are connected continuously, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that guarantee to be used for dyeing belong to same tangent planes.Two sections of same tangent plane are carried out respectively the detection of different testing proteins as a pair of.
A pair of paraffin section rinses through dewaxing, aquation, tap water and is placed in the EDTA solution (pH9.0) that is heated to boil 100 ℃ of laser heatings 20 minutes.Add the peroxidase blocking agent, its addition is wanted the ensuring coverage biopsy tissues, incubated at room 10 minutes, the PBS damping fluid (forms: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L, pH7.2) rinse 3 times each 3 minutes (namely rinsing 3 * 3 minutes).A slice add CD133 antibody (the mouse monoclonal antibody, 1:200), another section of a centering add acetaldehyde dehydrogenase antibody (the rabbit monoclonal antibody, 1:200), incubated at room 1 hour, PBS rinsed 3 * 3 minutes.Two anti-(the 50-200 μ L) that add the horseradish enzyme labeling, incubated at room 15 minutes, PBS rinsed 3 * 3 minutes.Add the DAB nitrite ion, incubated at room 5 minutes, tap water rinses color development stopping, and haematoxylin is redyed, and PBS returns indigo plant.(be 70% by volume ratio through gradient alcohol dehydration, 80%, 90%, 95%, 100% alcohol-pickled dehydration, wherein each concentration is 10 minutes) drying, dimethylbenzene soak 10 minutes transparent, then drip the neutral gum of 100-200 μ L on biopsy tissues, then careful covered, prevent from forming bubble.Carry out microscopic examination after the neutral gum mounting.Find out position corresponding to same cell, if same cell CD133, acetaldehyde dehydrogenase positive (wherein a cell in the tissue presents brown at two tissues of correspondence simultaneously) show in this tissue to have tumor stem cell.
The method of a kind of same many target point proteins of tangent plane immunofluorescence dyeing mark of the present invention comprises the steps:
The wax stone that will contain tumor tissues is fixed on histotome, tissue is cut into slices, when blade runs to destination organization, begin to choose, one serial section that is chained together is put into the ethanolic solution 1 minute to 5 minutes of 40%-60%, and then the hot water of putting into 40-60 ° of C is opened up sheet, choose two sections that are connected continuously, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that guarantee to be used for dyeing belong to same tangent planes.Two sections of same tangent plane are carried out respectively the detection of different testing proteins as a pair of.
A pair of paraffin section rinses through dewaxing, aquation, tap water and is placed in the EDTA solution (pH9.0) that is heated to boil 100 ℃ of laser heatings 20 minutes.The PBS(proportioning is with embodiment 1) rinse each 3 minutes (namely rinsing 3 * 3 minutes) 3 times.A slice adds CD24 antibody (the mouse monoclonal antibody of PE mark, 1:150), the CD44(rabbit monoclonal antibody of FITC mark, 1:200) mixed antibody, another section of one centering adds the ESA(1:200 of PE mark), the acetaldehyde dehydrogenase antibody (1:200) of FITC mark, incubated at room 1 hour, PBS rinsed 3 * 3 minutes.Carry out fluorescence microscope.Find out position corresponding to same cell, if the same cell of two tissues CD24, CD44, ESA, the aobvious green of acetaldehyde dehydrogenase and red fluorescence simultaneously show in this tissue to have tumor stem cell.
The present invention is described in detail below in conjunction with specific embodiment, and following examples are in order to further illustrate the present invention, but should not be considered as limiting the present invention.Below biochemical reagents used except indicating especially all available from Sangon Biotech (Shanghai) Co., Ltd., antibody and clinical tissue paraffin section are that applicant's (Foochow steps the neoformation technology development co.) provides, if operation steps is the routine operation step without explanation.
Embodiment 1(SABC 2 target spot marks):
The wax stone that will contain the liver neoplasm tissue is fixed on histotome, tissue is cut into slices, when blade runs to destination organization, begin to choose, a serial section that is chained together was put into 60% ethanolic solution 1 minute, and then the hot water of putting into 60 ° of C is opened up sheet, choose two sections that are connected continuously, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that guarantee to be used for dyeing belong to same tangent planes.Two sections of same tangent plane are carried out respectively the detection of different testing proteins as a pair of.
A pair of paraffin section rinses through dewaxing, aquation, tap water and is placed in the EDTA solution (pH9.0) that is heated to boil 100 ℃ of laser heatings 20 minutes.The peroxidase blocking agent that adds 150 μ L, incubated at room 10 minutes, the PBS damping fluid (forms: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 10mmol/L, KH2PO4 2mmol/L, pH7.2) rinse 3 times each 3 minutes (namely rinsing 3 * 3 minutes).A slice add CD133 antibody (the mouse monoclonal antibody, 1:200), another section of a centering add acetaldehyde dehydrogenase antibody (the rabbit monoclonal antibody, 1:200), incubated at room 1 hour, PBS rinsed 3 * 3 minutes.Add 200 μ L two of horseradish enzyme labeling anti-, incubated at room 15 minutes, PBS rinsed 3 * 3 minutes.Add the DAB nitrite ion, incubated at room 5 minutes, tap water rinses color development stopping, and haematoxylin is redyed, and PBS returns indigo plant.(be 70% by volume ratio through gradient alcohol dehydration, 80%, 90%, 95%, 100% alcohol-pickled dehydration, wherein each concentration is 10 minutes) drying, dimethylbenzene soak 10 minutes transparent, then drip the neutral gum of 100 μ L on biopsy tissues, then careful covered, prevent from forming bubble.Carry out microscopic examination after the neutral gum mounting.Find out position corresponding to same cell, if same cell CD133, acetaldehyde dehydrogenase aobvious positive (namely wherein a cell in tissue presents brown at two tissues of correspondence simultaneously) simultaneously show to have the liver tumour stem cell in this tissue.
Embodiment 2(SABC 4 target spot marks):
The wax stone that will contain pancreatic tumor tissue is fixed on histotome, tissue is cut into slices, when blade runs to destination organization, begin to choose, a serial section that is chained together was put into 40% ethanolic solution 5 minutes, and then the hot water of putting into 40 ° of C is opened up sheet, choose two sections that are connected continuously, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that guarantee to be used for dyeing belong to same tangent planes.Two sections of same tangent plane are carried out respectively the detection of different testing proteins as a pair of.
A pair of paraffin section rinses through dewaxing, aquation, tap water and is placed in the EDTA solution (pH9.0) that is heated to boil 100 ℃ of laser heatings 20 minutes.Add 160 μ L peroxidase blocking agents, incubated at room 10 minutes, the PBS(proportioning is with embodiment 1) rinse each 3 minutes (namely rinsing 3 * 3 minutes) 3 times.A slice add CD24 antibody (the mouse monoclonal antibody, 1:150), CD44(rabbit monoclonal antibody, 1:200) mixed antibody, another section of one centering adds ESA(mouse monoclonal antibody, 1:200), acetaldehyde dehydrogenase antibody (the rabbit monoclonal antibody, 1:200), incubated at room 1 hour, PBS rinsed 3 * 3 minutes.Add the anti-mouse two of horseradish enzyme labeling anti-rabbit two anti-and alkali phosphatase enzyme mark anti-, incubated at room 15 minutes, PBS rinsed 3 * 3 minutes.Add DAB, BCIP/NBT nitrite ion, incubated at room 5 minutes, tap water rinses color development stopping, and haematoxylin is redyed, and PBS returns indigo plant.(be 70% by volume ratio through gradient alcohol dehydration, 80%, 90%, 95%, 100% alcohol-pickled dehydration, wherein each concentration is 10 minutes) drying, dimethylbenzene soak 10 minutes transparent, then drip the neutral gum of 200 μ L on biopsy tissues, then careful covered, prevent from forming bubble.Carry out microscopic examination after the neutral gum mounting.Find out position corresponding to same cell, if same cell is CD24, CD44, ESA, acetaldehyde dehydrogenase aobvious positive (cell in namely wherein organizing presents brown and blue two kinds of colors simultaneously at two tissues of correspondence) simultaneously, show to have the pancreatic neoplasm stem cell in this tissue.
Embodiment 3(immunofluorescence 4 target spot marks):
The wax stone that will contain pancreatic tumor tissue is fixed on histotome, tissue is cut into slices, when blade runs to destination organization, begin to choose, a serial section that is chained together was put into 40% ethanolic solution 1 minute, and then put into 40 ° of C hot water and open up sheet, choose two sections that are connected continuously, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that guarantee to be used for dyeing belong to same tangent planes.Two sections of same tangent plane are carried out respectively the detection of different testing proteins as a pair of.
A pair of paraffin section rinses through dewaxing, aquation, tap water and is placed in the EDTA solution (pH9.0) that is heated to boil 100 ℃ of laser heatings 20 minutes.The PBS(proportioning is with embodiment 1) rinse each 3 minutes (namely rinsing 3 * 3 minutes) 3 times.A slice adds CD24 antibody (the mouse monoclonal antibody of PE mark, 1:150), the CD44(rabbit monoclonal antibody of FITC mark, 1:200) mixed antibody, another section of one centering adds the ESA(1:200 of PE mark), the acetaldehyde dehydrogenase antibody (1:200) of FITC mark, incubated at room 1 hour, PBS rinsed 3 * 3 minutes.Carry out fluorescence microscope.Find out position corresponding to same cell, if the same cell of two tissues CD24, CD44, ESA, the aobvious green of acetaldehyde dehydrogenase and red fluorescence simultaneously show to have the pancreatic neoplasm stem cell in this tissue.
Embodiment 4:
To contain the wax stone that brain tumor knits is fixed on histotome, tissue is cut into slices, when blade runs to destination organization, begin to choose, a serial section that is chained together was put into 60% ethanolic solution 5 minutes, and then the hot water of putting into 60 ° of C is opened up sheet, choose two sections that are connected continuously, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that guarantee to be used for dyeing belong to same tangent planes.Two sections of same tangent plane are carried out respectively the detection of different testing proteins as a pair of.
A pair of paraffin section rinses through dewaxing, aquation, tap water and is placed in the EDTA solution (pH9.0) that is heated to boil 100 ℃ of laser heatings 20 minutes.The PBS(proportioning is with embodiment 1) rinse each 3 minutes (namely rinsing 3 * 3 minutes) 3 times.A slice adds the mixed antibody of anti-musashi-1 monoclonal antibody (1:200) of Sox-2 monoclonal antibody (1:200), the AMCA mark of CD133 antibody (1:200), the FITC mark of PE mark; Another sheet adds the mixed antibody of anti-melk monoclonal antibody (1:200) of nestin monoclonal antibody (1:200), the AMCA mark of Bmi-1 antibody (1:200), the FITC mark of PE mark, incubated at room 1 hour, and PBS rinsed 3 * 3 minutes.Carry out fluorescence microscope.Find out position corresponding to same cell, if the same cell of two tissues aobvious green, the redness of CD133, Sox-2, musashi-1, Bmi-1, nestin, melk and blue-fluorescence simultaneously show to have the brain tissue tumor stem cell in this tissue.
Claims (5)
1. the method for same many target point proteins of tangent plane SABC or immunofluorescence dyeing mark, it is characterized in that: when paraffin embedded tissues is cut into slices, get two continuous sections, when adopting microslide to drag for sheet, the back side of first section up, be right after second and face up when dragging for sheet, two histotomies surfaces that namely are used for dyeing belong to same tangent planes.
2. the method for a kind of same many target point proteins of tangent plane SABC according to claim 1 or immunofluorescence dyeing mark, it is characterized in that: to belonging to two sections of same tangent plane, carry out respectively SABC or the immunofluorescence dyeing of different target point proteins, during observation, the tissue staining result of getting corresponding the same area overlaps comparison, realizes the existence analysis of the many target point proteins in same cell or tissue site.
3. the method for a kind of same many target point proteins of tangent plane SABC according to claim 1 or immunofluorescence dyeing mark, it is characterized in that: when described two continuous sections refer to histotomy, be close to two sections together, the front of the back side of first section and second section is the two sides of the same tissue after single solution for diverse problems is opened.
4. the method for a kind of same many target point proteins of tangent plane SABC according to claim 1 or immunofluorescence dyeing mark, it is characterized in that: when the front of described section refers to cut into slices, in the face of tangent plane operator's one side, the described back side is near the one side of tissue back to the operator.
5. the method for a kind of same many target point proteins of tangent plane SABC according to claim 2 or immunofluorescence dyeing mark is characterized in that: described target point protein refers to have in pathology detection the target protein of diagnosis effect.
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| CN113567482A (en) * | 2021-07-16 | 2021-10-29 | 江南大学 | Method for monitoring internal component structure and distribution of grains in cooking process |
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