108 results about "DAPI" patented technology

The invention relates to an immunofluorescence staining method for suspension cells. The immunofluorescence staining method for the suspension cells is characterized by comprising the following steps of: collecting cells into a centrifuge tube; performing 800-gram centrifugation to collect the cells; respectively adding paraformaldehyde to fix, Triton-X100 to pass through and 1 percent bovine serum albumin (BSA) to close; adding a primary antibody and a secondary antibody sequentially to incubate; washing by an 800-gram centrifugation method after the operation of each step; performing diamino phenyl indole (DAPI) staining; and dripping on a glass slide and closing the glass slide to observe. The immunofluorescence staining method for the suspension cells has the advantages that: the immunofluorescence staining process is performed in the centrifuge tube, so the problems that the suspension cells grow on the glass slide difficultly and drop off easily in the test process are solved; the staining result is good; and the method is suitable for the suspension cells and cells which are adhered to the wall difficultly.

The invention relates to a preparation method for probes related to breast cancer molecular markers and to preparation of a breast cancer fluorescence in situ hybridization detection kit by using the probes. The breast cancer fluorescence in situ hybridization detection kit can be prepared from HER2, TOP2A and AGTR1 gene probes prepared by using the method provided in the invention, human chromosome 17 counting probe, hybridization buffer, unlabelled competitive DNA and DAPI counter strain; application of the kit enables the detection rate of breast cancers to be substantially improved and type sorting of breast cancers to be more accurate and provides guidance to formulation of an individual therapeutic schedule and selection of proper therapeutic drugs, thereby lowering down mortality, reducing recurrence risk and achieving the goal of optimizing the effect of diagnosis and treatment.

The invention discloses a detecting method for a circulating tumor cell surface marker molecule PD-L1. The detecting method comprises the following steps: (1) treating whole blood with red blood celllysate to separate out karyocyte, and fixing with formaldehyde; (2) performing positive screening through tumor immune fluorescent marker cytokeratin antibody anti-CK, performing PD-L1 primary antibody incubation on all cells, and performing PD-L1 secondary antibody incubation with a fluorescence group marked with FITC, and marking all cells through cell nucleus fluorescent dye DAPI; and (3) adopting high-flux multi-color imaging analysis to select light filter plates of CY5, FITC and DAPI, observing fluorescent color on the surface of a channel, and finally realizing detection on the circulating tumor cell surface marker molecule PD-L1. The detecting method is simple and convenient, is quick, is economical, is non-invasive, is high in sensitivity and is good in specificity.

A method of utilizing nuclear specific fluorescent staining and ovary being made to be completely transparent technique to observe blastocyst of rice includes using certain concentration of sodium hydroxide solution to carry out softening treatment on blastocyst of rice before staining then utilizing specific combination of nucleus fluorescent staining DAPI with nucleus to observe structure of cell and nucleus in blastocyst, enabling to use laser scan confocal microscope to observe internal structure of cell clearly.

The invention discloses a floating sphere immunofluorescent staining method and a staining device, belonging to methods for coloring a sample for test. The staining method comprises the following steps: separation of spheres from a culture medium, fixing by stationary liquid, adding of Triton X-100 transparent cells, closing by confining liquid, adding of an interest protein-resisting primary antibody working solution, adding of a primary antibody-resisting fluorescent secondary antibody working solution, and cell nucleus staining by DAPI dye liquor. The staining device comprises a cell chamber and a staining sleeve part arranged at the outer part of the lower end of the cell chamber in a sleeving way. The floating sphere immunofluorescent staining method has the advantages of high efficiency, simpleness, convenience and the like, an antibody usage amount is reduced, experiment cost is reduced, manpower and time are saved, an accurate and reliable experiment result is obtained on the basis that a fundamental principle that immunofluorescent staining is not changed and specially training experimenters is not needed, sphere loss in an experiment operation process is avoided, an experiment success rate is increased, and the efficiency is improved.

The invention discloses a differential staining method for inner cell mass cells and trophoblastic cells of cattle blastulae. The method comprises the following steps: 1, carrying out immune combination on an anti-CDX2 monoclonal antibody which is adopted as a primary antibody and CDX2 molecules which are specifically expressed in the trophoblastic cells of the cattle blastulae through an immunostaining process; 2, the cattle blastulae which are cleaned and are combined with the primary antibody are immunostained with an antibody which is labeled by red fluorescence and can combine with the anti-CDX2 monoclonal antibody as a secondary antibody through the immunostaining process; and 3, whole nuclei of the cattle blastulae are subjected to fluorescent staining after completing the immunostaining to form the differential staining. On the basis of a case that CDX2 proteins specifically express in the trophoblastic cells and do not express in the inner cell mass cells, the method of the invention allows the differential staining to be carried out based on the red immunostaining of the CDX2 molecules and the blue staining of DAPI nuclei, so the accuracy is 100%, and obtained pictures are intelligible and beautiful.

The invention discloses a DNA (deoxyribonucleic acid) dye compound with a nuclear targeting function and an application, and belongs to the field of fine chemical engineering. In the field of bioimaging, visible research of genetic materials is of great significance for cellular activity research. The wavelength of exciting light needed by dyes Hoechst and DAPI of traditional targeting cell nucleuses is short, and serious cellular damage is caused in imaging. In addition, the Hoechst and the DAPI can only purely target the cell nucleuses, but cannot achieve other functions. In order not to affect normal physiological functions of cells, the DNA dye compound has high biocompatibility, can be specifically combined with DNA and has the nuclear targeting function.

The invention belongs to the field of biomedicine clinical detection, and particularly relates to a kit applying CD45 immunofluorescence combined with a CEP fluorescent in-situ hybridization probe to identify circulating tumor cells and application thereof. The kit comprises fixing liquid, 2xSSC, 0.3%NP-40 / 0.4xSSC, 0.075M KCI solution, CD45 monoclonal antibody and CEP probe mixed liquid, DAPI counter stain, perforating agent, closing liquid and mounting medium. The CD45 immunofluorescence antibody is combined with the CEP fluorescent in-situ hybridization probe to identify captured circulating tumor cells, and immunofluorescence and fluorescent in-situ hybridization detection are completed through a one-step process, so that the problems of mutual interference and long detection time when immunofluorescence detection and fluorescent in-situ hybridization detection are combined are solved; hybridization incubation through the one-step process is completed quickly within 2h, so that detection time is greatly reduced, and detection results are visual to observe.

The invention relates to a DNA nano-origami structure constructed by rolling circle amplification technology and DNA origami, at the same time utilizes fluorescent dye DAPI with DNA double-strand binding properties and cell membrane penetration properties to label and track the DNA nano-origami structure, thus realizing real-time monitoring and simulation of the drug loading and sustained release process of the DNA nano-origami structure as a drug carrier. And the method also can be applied as a new fluorescent quantitative analysis method to biomolecular detection, like tumor early detection, postoperative monitoring and evaluation, cell imaging and other fields.

The invention belongs to the field of biomedical clinical detection and relates to a kit for identification of circulating tumor cells through CD45 immunofluorescence-CEP17 fluorescence in-situ hybridization probe combined one-step method co-dyeing, and a use thereof. The kit comprises a fixing solution, 2*SSC, 0.3% NP-40 / 0.4*SSC, 0.075 M of a KCl solution, a CD45 monoclonal antibody-CEP17 probe mixed liquid, a DAPI redyeing agent, a perforating agent, a sealing liquid and a mounting medium. The kit uses combination of a CD45 immunofluorescent antibody and a CEP17 fluorescence in-situ hybridization probe to identify the captured circulating tumor cells, completes immunofluorescence and fluorescence in-situ hybridization detection by a one-step method, and solves the problem that the immunofluorescence detection and the fluorescence in-situ hybridization detection produce mutual interference in combined use and detection time is long. The one-step method-based hybridization incubation can be fast finished in 2h so that detection time is greatly reduced. The detection result is visual.

The invention discloses a method for separating a cell nucleus from a human frozen tumor tissue suitable for single cell sequencing and a method for extracting the cell nucleus. The method for extracting the cell nucleus comprises performing splitting treatment on a sample to be extracted in a first buffer solution; performing first centrifugation on a split product to obtain a cell nucleus precipitate; rinsing the cell nucleus and conducting resuspending treatment in a second buffer solution to obtain the cell nucleus; wherein the first buffer solution comprises: 116.8 mM of NaCl, 8 mM of Tris base at pH 7.8, 0.8 mM of CaCl2, 38 mM of MgCl2, 0.04% BSA, 0.16% Nonidet P-40, 1 mM of EDTA and 1 mg / mL of DAPI. The second buffer solution comprises 1*PBS, 1.0% BSA and 0.2U / [mu]L of RNase inhibitor. According to the method, only a simple reagent is required, no expensive separation equipment is required, and sufficient complete mononuclear can be obtained from the less frozen tumor tissue forsingle cell RNA sequencing.

The invention discloses a database establishing method suitable for plant transposase accessible chromatin analysis. The database establishing method comprises following steps: firstly, plant tender tissue is subjected to pre-treatment, enzymatic hydrolysis is cell walls is carried out, filtering is carried out, DAPI dyeing is adopted to sort and collect nucleuses on a flow cytometer, the obtainednucleuses are filtered so as to reduce interference of cell walls and subcellular organelles on experiment data; Tn5 transposase digestion reaction and purification are carried out, qPCR is adopted to determine circulating number needed by database establishing; at last obtained single library is subjected to equimolar mixing. According to the database establishing method, the high quality on computer library is obtained via optimized two circles of magnetic cell sorting at a certain ratio, so that important experiment method reference is provided for study of scientific research personnel onplant tender tissue accessible transposase nucleus chromatin high flux sequencing.

A method for stably labeling liver cancer cell includes combining liver cancer cell with specific AFP single cloning antibody used as the first resistance factor, washing and climbing plate of PBS, carrying out fluorescent label by using QDs ¿C IgG composite probe as the second resistance factor, washing and climbing plate of PBS, re ¿C staining cell core with DAPI, washing and climbing plate of PBS and preserving sample in light shielded condition.

The invention discloses membrane permeability dye with a large two-photon fluorescence active cross section. The dye adopts triphenylamine heterocyclic chemical compounds, and the chemical structure of the dye is shown in the formula (I). The invention further discloses an application of the dye in displaying two-photon imaging of cytoplasm in a living cell. Experiments show that the dye has characteristics of larger two-photon fluorescence active absorption cross section, excellent cell membrane permeability, low toxicity and the like, also has the characteristics of wide application range, low price and good bio-compatibility with known probe DAPI (4',6-diamidino-2-phenylindole) and has wide application prospect in the field of laser excitation fluorescence biomarkers.

A biopsy-sized tissue sample is stained for quick imaging. A significant amount of permeation enhancer is included in a mixed solution of permeant enhancer, fixative or dehydrant, and one or two fluorescent dyes to simultaneously dehydrate and dye the tissue sample. The permeation enhancer, e.g., 10% to 50% in the mixed solution, achieves an image of dyed tissue in the contacted tissue sample at a depth of at least 200 um within no more than 1.5 hours. One of the fluorescent dyes is a fluorescent nuclear dye such as DAPI, SYTOX green, acridine orange, propidium iodide, or a Hoechst dye. The other fluorescent dye is a fluorescent protein dye such as eosin or rhodamine B. The tissue sample is cleared with a clearing agent having a refractive index of at least 1.4[R2], e.g., using BABB. The mixed solution may further include Chloroform or other morphology preservative.

The invention relates to a novel high-resolution quantitative multicolor fluorescent in-situ hybridization method and application thereof. The method provided by the invention is implemented in a way that: a fluorescein label BAC (bacterial artificial chromosome) probe is adopted, and a continuous fluorescent in-situ hybridization method (probe elution rehybridization) is utilized; and after the hybridization each time, fluorescent information in six optical filter channels DAPI, Spectrum GreenTM, Cy3TM v1, Texas Red, Cy5 and PF-415 are acquired under a fluorescent microscope, and a computer automatic fluorescent visual field relocation method is utilized to accurately locate all the acquired images for all slices to the same visual field, thereby simultaneously analyzing 5-20 genes of a single normal or tumor cell. The method provided by the invention can screen and verify multiple gene mutants on the unicell level, and can be used for distinguishing different subtypes of multiple diseases as well as further classification of the same subtype of disease.

The invention discloses a fluorescence labeling method for detecting DNA matters of a cotton pollen mother cell, and belongs to the biological technical field. The method comprises the following steps: on a sunny morning, buds are picked up, and a sample passes through ethanol with reducing concentrations to enter distilled water; pollen mother cells with a diameter of less than 7mm of the buds are placed in glycerin for infiltration for 2 to 3 hours, and, after washing, are dyed by DAPI in the dark for 1 to 2 hours to label DNA matters in the cells; mature pollen grains with the diameter of more than 7mm of the buds are added with a 10 percent sodium hypochlorite aqueous solution, and subjected to water bath treatment at a temperature of 60 DEG C for 10 minutes, washed, placed in 0.1 to 0.2 percent glycerin for infiltration for 2 to 3 hours, washed, added with DAPI solution, stood for 10 minutes and subjected to microscopic examination to label DNA matters in the cells. The method has the advantages of simple and quick operation, less damages to cells, good labeling effect and easy observation, and can play an important role in research on the development process of cotton male gametophyte, and has high application value.

The invention provides an immunofluorecence technique based method for evaluating activities of cryptosporidium parvum and giardia. The method has the following beneficial effects: the accurate, quick, simple, convenient and low-cost high throughput immunofluorecence assay technique is established and is used for evaluating the activities of cryptosporidium parvum and giardia in the ultraviolet (UV) disinfectant fluid; evaluation of the activities of cryptosporidium parvum and giardia in the UV irradiated water is realized by mainly carrying out competitive immunobinding on anti-pyrimidine-dimer (TDs) monoclonal antibodies and the TDs, taking fluorescein isothiocyanate as a marker and immunoglobulin (IgG) as a fluorescent substrate and binding 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI); compared with the fluorescent staining method for evaluating the effect of UV on inactivating cryptosporidium parvum and giardia, the method provided by the invention has such excellent characteristics as strong specificity, high sensitivity and low professional operation requirements; and the cryptosporidium parvum and giardia activity evaluation method system is perfected by binding the method provided by the invention with the fluorescent staining method and other methods, and the method provided by the invention can be widely applied to the fields such as water quality monitoring, disease control and epidemic prevention and the like.

The present invention is a method for detecting or quantifying a target cell or a target protein using an aptamer chip, and particularly, in order to detect a target cell, a method for detecting and / or quantifying a target cell by reacting a cell staining solution (e.g. 4′,6-diamidino-2-phenylindole (DAPI)) with an aptamer chip which the target cell is bound, in order to detect a target protein, a method for detecting and / or quantifying a target protein by reacting Coomassie Brilliant Blue solution with an aptamer chip which the target protein is bound, and a method for reusing the aptamer chip, wherein the aptamer chip comprises a board to which the aptamer specifically combining with the target cell or the target protein is bonded by a disulfide bond.

The invention discloses a method for analyzing the cycle G1, the cycle S and the cycle G2 / M of wheat root tip cells through flow cytometry and belongs to the technical field of biology.The method comprises the steps of immersing wheat seeds and accelerating germination, obtaining a wheat root tip sample through cutting, and fixing the sampling with Carnoy fixing liquid; rinsing the sample thoroughly with a phosphate buffer solution; placing the sample in a mixed enzyme liquid of cellulose and pectinase for conducting enzymolysis for 1-2 hours; after the sample is rinsed thoroughly, conducting grinding, so that single-cell suspension is obtained; after rinsing is conducted, conducting dyeing away from light on the wheat root tip cells through DAPI dye liquor; after rinsing and filtering are conducted, differentiating and counting the cells of cycle time phases including the cycle G1, the cycle S and the cycle G2 / M which are different in DNA content through a flow cytometer, and learning about the proliferation condition of the cells.The method is simple and easy to operate, separated materials are easy to obtain, the enzymolysis method is simple, the obtained single-cell suspension is high in purity, the flow cytometer is good in detection effect, and the result is stable.

The invention relates to a single wavelength excited double signal-enhanced Hg<2+> fluorescent ratio method and belongs to the technical field of chemical / biological sensing and analysis detection. The method comprises adding a single-stranded DNA molecule A, a single-stranded DNA molecule B, NMM and DAPI into a buffer solution containing Mg<2+> to obtain a mixed solution I, wherein the molecule Acomprises a part I and a part II, the molecule B comprises a part III and a part IV, the part I in the molecule A and the part III in the molecule B are partially complementary sequences with T-T basic group mismatching and the part II in the molecule A and the part IV in the molecule B form a four-stranded G-rich sequence, adding Hg<2+> into the mixed solution I, and carrying out vibration and measuring fluorescence intensity in a measuring range of 400-700nm and excitation wavelength of 380nm. The method is a double signal-enhanced novel Hg<2+> ratio-type fluorescence analysis method, is used for detection of Hg<2+> in a sample and has high selectivity, accuracy and reliability.

The invention provides an immunofluorescence kit and method for E-Cadherin mutation of peripheral blood circulating tumor cells of a non-small cell lung cancer patient, and belongs to the technical field of molecular biology. The kit comprises 45 mL of a diluent, 1 mL of a decolorizing solution, 0.5 mL of a staining solution A, 1 mL of a staining solution B, 200 [mu] l of methanol, 200 [mu] l of 2% PFA, 100 [mu] l of 10% goat serum, 100 [mu] l of a primary antibody working solution, 100 [mu] l of a secondary antibody working solution and a DAPI mounting medium. According to the detection method provided by the invention, the E-Cadherin expression condition of the patient suffering from advanced or recurrent non-small cell lung cancer can be detected without obtaining a tissue specimen through needle biopsy. The technology belongs to minimally invasive technology and can realize real-time detection. According to the method provided by the invention, a false positive result caused by anedge effect possibly generated in the dyeing process can be avoided, the stability is high, the cell loss is reduced, and the detection accuracy is improved.

The invention provides an immunofluorescence kit and a detection method for detecting PD-L1 gene mutation of peripheral blood circulating tumor cells of a small cell lung cancer patient, and belongs to the technical field of molecular biology. The kit comprises 45 mL of a diluent, 1 mL of a decolorizing solution, 0.5 mL of a staining solution A, 1 mL of a staining solution B, 200 [mu] l of methanol, 200 [mu] l of 2% PFA, 100 [mu] l of 10% goat serum, 100 [mu] l of a primary antibody working solution, 100 [mu] l of a secondary antibody working solution and a DAPI mounting medium. According to the detection method provided by the invention, the PD-L1 expression condition of a patient suffering from advanced or recurrent small cell lung cancer can be detected without obtaining a tissue specimen through needle biopsy. The technology belongs to minimally invasive technology and can realize real-time detection. According to the method provided by the invention, a false positive result causedby an edge effect possibly generated in the dyeing process can be avoided, the stability is high, the cell loss is reduced, and the detection accuracy is improved.

The present invention provides an immunofluorescent double staining kit for cervical cancer auxiliary diagnosis. The kit comprises a mixed primary antibody working solution consisting of a glucan labeled Ki67 monoclonal antibody and a glucan labeled P16INK4A monoclonal antibody, and a DAPI staining solution. The kit simultaneously and efficiently excites multi-color fluorescent light, and all colors can be showed by only one kind of excitation light, so that wavebands do not need to be switched and an ordinary microscope is directly used for observation and shooting; a secondary antibody incubation process is removed, thereby shortening the operation time of the whole process; and the operation is simple, and the sensitivity and the accuracy are high.

The invention discloses an immunofluorescent method for detecting plant gibberellin, and belongs to the field of plant biotechnology. The immunofluorescent method for detecting plant gibberellin comprises the following steps: S1, dewaxing rehydration; S2, antigen retrieval; S3, autofluorescence quenching; S4, serum sealing; S5, addition of one antibody; S6, addition of two antibodies; S7, DAPI cell nucleus redyeing; S8, mounting; and S9, microscopic examination. The method is simple, rapid, safe and non-toxic, the detection cost is reduced effectively, histocytes are positioned by using DAPI redyeing cell nucleuses, and the position of antigen distribution can be observed and determined preferably; the distribution condition of gibberellin inside plant tissues can be observed through a fluorescence microscope after dyeing; the original states of plant tissues and cells are maintained to the maximum extent in the experimental process; and the biological activity and immunocompetence canbe maintained after marking.

The invention discloses a cancer screening and diagnosis method via staining quantification of cellular DNA by a fluorescent dye DAPI. According to the method, cell nucleuses in cell smears or cells and tissue sections are stained with DAPI as a dye, pictures are taken under a fluorescence microscope, and the DNA in each nucleus is relatively quantified according to the fluorescence intensity of each nucleus. Cells with abnormal DNA content (non-integer ploidy or ploidy number being greater than 4.5 or above) are suspected cancer cells. The number and morphology of the suspected cancer cells can be used for cancer screening and diagnosis. The dyeing method provided by the invention has the advantages of short dyeing time, simple operation and accurate quantification, is wide in application, can quantify the DNA of cells in cell smears, tissue sections and cultured cells, and can be used for screening cancer samples.

The invention belongs to the technical field of molecular biology, and particularly relates to an immunofluorescence kit for detecting E-Cadherin expression of peripheral blood circulating tumor cellsof a patient with renal cell carcinoma, and a detection method. The immunofluorescence kit comprises a diluents 45 mL, decolorizing liquid 1 mL, a staining solution A 0.5 mL, a staining solution B 1mL, methanol 200 mu l, 2% PFA 200 mu l, 10% goat serum 100 mu l, a primary antibody suspension 100 mu l composed of mouse anti-CK, rat anti-CD45 and rabbit anti-E-Cadherin, a secondary antibody suspension 100 mu l composed of fluorescently-labeled goat anti-mouse, fluorescently-labeled goat anti-rat and fluorescently-labeled goat anti-rabbit, and a DAPI tablet. According to the detection method provided by the invention, the E-Cadherin expression condition of the patient with later-period or relapse renal cell carcinoma can be detected without acquiring a tissue specimen through needle biopsy.The immunofluorescence kit belongs to minimally invasive technology and can realize real-time detection.

The invention belongs to the technical field of molecular biology, and particularly relates to an immunofluorescence kit for detecting PD-L1 expression of peripheral blood circulating tumor cells of akidney cancer patient and a detection method. The kit comprises 45mL of a diluent, 1mL of a destaining solution, 0.5mL of staining solution A, 1mL of a staining solution B, 200[mu]l of methanol, 200[mu]l of 2% PFA, 100[mu]l of 10% goat serum, 100[mu]l of a primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1, 100[mu]l of a secondary antibody suspension composed of a fluorescently labeled goat anti-mouse, a fluorescently labeled goat anti-rat and a fluorescently labeled goat anti-rabbit, and a DAPI mounting medium. According to the detection method provided by the invention, the PD-L1 expression condition of a patient with advanced or recurrent renal cancer can be detected without acquiring a tissue specimen through needle biopsy. The technology belongs to minimally invasive and can realize real-time detection.

The invention discloses a method for detecting reactive oxygen content in living cells through DAPI fluorescent dye. According to the method, the DCF fluorescence signal values of the single living cells corresponding to exposed contaminants at different concentrations can be obtained. According to the method for detecting the reactive oxygen content in the living cells, the detecting result is precise, the problem that due to the fact that the contaminants are exposed, the number of the cells changes, and final fluorescence intensity measurement is inaccurate is solved, the fluorescence signal values of the single living cells corresponding to a certain concentration can be truly reflected through the method for detecting the reactive oxygen content, therefore, the damage of contaminant exposure to the cells can be truly and accurately reflected, and a more solid foundation is laid for application of an in-vitro cell model in contaminant toxicity assessment.