83 results about "Immunofluorescence staining" patented technology

The invention discloses a method and an automatic instrument device for enriching and extracting target cells and separating single cells by using a positive magnetic bead method and performing immunity and molecular biology identification and analysis on single cells. By the method and the instrument device, the on/off of a capture magnet and a release magnet is controlled by various methods to complete target cell searching, capturing, cleaning and releasing operation once or for multiple times, so that the target cell detection sensitivity and stability are improved. When the capture magnet searches and captures the target cells, the search line is circular, square, comb-shaped, S-shaped or U-shaped. In addition, the captured substances are filtered, most free micro magnetic beads are removed, the purity of the product is further improved and the product can be used as a good experimental material. By combining a special filter and the adsorption of the capture magnet, more than 95 percent of free micro magnetic beads can be effectively removed. Meanwhile, the types of the single cells are identified and biological characteristics of the single cells are analyzed by an immunofluorescence staining method and a method in molecular biology, and effective biological indexes are provided for clinical diagnosis and treatment of cancers.

The invention relates to medicine application of 20(S)-ginsenoside Rg3, in particular to application of 20(S)-ginsenoside Rg3 in preparation of medicines for treating a non-small cell lung cancer. In the invention, selecting a human non-small cell lung cancer cell strain (A549 lung adenocarcinoma, H460 large cell lung cancer and LTEP-78 lung squamous carcinoma) to be inoculated under the skin of a naked mouse, and observing the influence of the SPG-Rg3 on the non-small cell lung cancer; and carrying out CD34 immunohistochemistry staining and TUNEL immunofluorescence staining on tumor tissues, and observing the influence of the SPG-Rg3 on tumor angiogenesis and apoptosis. The inclusion proves that the 20(S)-ginsenoside Rg3 can remarkably inhibit the growth of the non-small cell lung cancer, has the action mechanism related with the promotion of the tumor apoptosis and the inhibition of the tumor angiogenesis and unobvious cooperation action when being combined with cyclophosphamide for application, and can be used for the clinical chemotherapy of the non-small cell lung cancer.

The invention discloses a lentiviral plasmid expression vector as well as a construction method and an application of the lentiviral plasmid expression vector. The lentiviral plasmid expression vector is pCDH-MCS-MuSK-GFP and the nucleotide sequence of the lentiviral plasmid expression vector is shown in SEQ ID NO. 2 in a sequence table. The invention further discloses an application of the lentiviral plasmid expression vector in construction of an anti-muscle-specific receptor tyrosine kinase antibody detection. According to the invention, a stable transfected cell line of MuSK is established by constructing a viral vector, a MuSK antibody is qualitatively and quantitatively detected clinically by an immunofluorescence staining method with relatively high sensitivity and specificity in combination with a flow cytometry, the detection results are more stable and manpower and time costs are greatly saved. The diagnosis and treatment levels of myasthenia gravis are greatly improved by the establishment of the project, the labor capacity and life quality of patients are improved and the lentiviral plasmid expression vector has significant social and economic benefits.

The invention discloses a method and a device for inducing bone marrow mesenchymal stem cell differentiation and proliferation, and belongs to the technical field of biological cell engineering neural re-restoration. The method comprises the following steps of: (1) taking two conducting copper wires, fixing the two conducting copper wires on the two ends of a conducting glass conducting surface, and performing sterilization treatment for use after the fixation; (2) taking a fresh isolated humerus, cleaning the fresh isolated humerus, then, sucking bone marrow mesenchymal stem cells into a centrifugal tube, removing liquid supernatant through centrifugation, adding the cells into a culture medium to be uniformly blown and beaten, and then inducing the cells into a culture bottle to be culture for 3 to 5 generations for use; and (3) inducing the cells cultured in the second step onto conducting glass processed by the first step, performing electrical stimulation on the cells after the culture, and obtaining the differentiation result of the cells in the direction towards neurons through the immunofluorescence staining and the gene expression detection. The low-frequency alternating current electricity harmless on organisms and cells is used; a physical physical stimulation method is used for stimulating the stem cells, so that the stem cells can differentiate towards the neurons; the operation of the method is easy; the electric stimulation on be given at any time; and high controllability is realized.

The invention relates to a double-drug loaded nanoparticle and a preparation method and application thereof. Being a water-in-oil-in-water nanomicelle, the double-drug loaded nanoparticle comprising amphiphilic polymers PEG-PPS (polyethylene glycol-polyphenylene sulfite), hydrophobic drugs and hydrophilic drugs is prepared through the emulsification reaction of the amphiphilic polymers, hydrophobic drugs and hydrophilic drugs. The preparation method of the double-drug loaded nanoparticle is simple and easy to apply and solving the problem of compounding the hydrophilic drugs and hydrophobic drugs in a nano drug delivery system. Furthermore, the double-drug loaded nanoparticle prepared through the preparation method has the advantages of passing various detection such as distribution detection of nano-components in vivo, immunofluorescence staining of tumor tissues, detection of ATP (adenosine triphosphate) content, detection of ROS (reactive oxygen species) in vivo and anti-tumor experiments and exhibiting a good photodynamic therapy effect.

The invention discloses a method for detecting rare cells in blood based on surface enhancement effect, which is characterized by comprising the steps of: plating a layer of plasma colored metal membrane on the surface of slide using spray coating method, and obtaining a SERS chip with metal nanostructure; arranging the SERS chip into a capture box of a capture apparatus; loading a pretreated blood sample in the capture apparatus, setting operating parameters of the capture apparatus, and capturing rare cells in the blood sample by an immune-magnetic enrichment method; taking the SERS chip with metal nanostructure out of the capture box after capturing, and performing immunofluorescence staining on the SERS chip after drying at 37 DEG C; and scanning and detecting the stained rare cells using a detection system. According to the method for detecting rare cells in blood based on surface enhancement effect, the capturing efficiency of rare cells such as CTC cells in the blood can be obviously improved, and the detection signal of rare cells can be enhanced

The invention discloses a production method of a skin pathological section. The method comprises the following steps: obtaining a material from a living body and fixing in-vitro skin tissues; dehydrating in-vitro tissues, immersing with wax, embedding, sliding and dyeing to prepare the skin pathological section. Material taking, fixing and embedding methods of animal skin and subcutaneous tissuesare standardized and the problems that the consistency of a skin pathological specimen and a living body tissue shape is poor and information of a pathological picture is not complete are solved; under the condition that the operation difficulty is not remarkably improved, self control also can be provided for skin immunohistochemistry and immunofluorescence staining.

The invention relates to an immunofluorescence staining method of paraffin embedded three-dimensional suspension cell clusters. The method comprises the following steps: pretreating the three-dimensional suspension cell clusters, immersing into paraffin liquid to embed sections, carrying out antigen repair, and carrying out immunofluorescence staining. According to the method provided by the invention, cells in the cell cluster are fully dyed, the result is accurate, the overall protein expression and distribution condition of the suspension cell cluster can be observed, and sample observationcan be carried out only by a common fluorescence microscope.

The invention provides a method for detecting the expression quantity of HOXD9 after peripheral nerve injury. The method comprises the following steps: (1) separating dorsal root ganglion neuron nucleoproteins at different time points after sciatic nerve injury of a rat, performing transcription factor activity detection, and determining the expression quantity change trend of a transcription factor HOXD9 in the nerve regeneration process; (2) observing the expression quantity and the expression trend of related messenger RNA results of HOXD9 by applying a PCR technology; (3) displaying intracellular localization of the HOXD9 in an DRG neuron by adopting an immunofluorescence staining technology; and (4) counting the HOXD9 fluorescence intensity change trends at different time points afterthe sciatic nerve injury, and enabling the fluorescence intensity to be the highest on the third day and to be consistent with a result trend obtained by a PCR experiment. The expression quantity change of HOXD9 after peripheral nerve injury is clarified, and the regulation effect of THE HOXD9 on neuron axon regeneration is discovered.

The invention discloses a method for detecting expression distribution level of GAPDH protein in bone marrow mesenchymal stem cells, and the application of the method in authenticating the activity of the bone marrow mesenchymal stem cells. The invention also discloses a method for authenticating the activity of the bone marrow mesenchymal stem cells. The method comprises the following steps: (1) detecting the expression distribution level of the GAPDH protein in the bone marrow mesenchymal stem cells by adopting a cell immunofluorescence staining method; (2) if over 60 percent of the GAPDH protein is distributed in cell nuclei, determining that the GAPDH protein is of a highly expressed type and the activity of the bone marrow mesenchymal stem cells is high; if less than 40 percent of the GAPDH protein is distributed in cell nuclei, determining that the GAPDH protein is of a lowly expressed type and the activity of the bone marrow mesenchymal stem cells is low. According to the method, the relationship between the expression distribution level of the GAPDH protein in the bone marrow mesenchymal stem cells and the activity of the bone marrow mesenchymal stem cells is discovered for the first time; by the provided method, the bone marrow mesenchymal stem cells which have a good treatment effect on diseases and high multiplication capacity can be authenticated quickly, so that the method can be used for high-flux screening of the bone marrow mesenchymal stem cells.

The invention discloses an immunofluorescence staining method for buffalo spermatogonial stem cell-like cells, comprising the following steps: (1) suspension preparation: cutting buffalo testicles into pieces, performing two-step digestion, using a pipette for pipetting to obtain a single cell suspension which is then resuspended and filtered; (2) cell purification: inoculating the suspension intoa culture dish for culturing, collecting suspended and non-adherent cells, and collecting adherent cells after culturing for two times to obtain purified spermatogonial stem cell-like cells; (3) cellfixation: fixing the cells with a tissue fixing solution, spraying the fixed spermatogonial stem cell-like cells on a slide at a dose of 5[Mu]l/drop and drying; (4) incubation: drawing a circle around a specimen with a pap pen, performing permeable membrane positioning on genes in a cytoplasm or a nucleus, and incubating and washing with a primary antibody and a secondary antibody; and (5) staining: dripping a staining agent to the immunohistochemical circle, and putting a coverslip on the circle for sealing with a nail enamel seal. The immunofluorescence staining method for buffalo spermatogonial stem cell-like cells provided by the invention is easy to operate and has a good staining effect.

The invention discloses a method for obtaining a treatment target causing skin keloids, and relates to the field of biomedical engineering and proteomics, and the method comprises the following steps:firstly, obtaining a skin matrix scaffold of human keloids; identifying the matrix protein component of the skin keloid skin by using tandem mass spectrometry; then comparing the keloid with matrix protein components of normal skin, and screening a keloid diagnostic marker; then carrying out immunofluorescence staining to confirm the specific mechanism of keloid formation; and finally, screeninghormone receptors serving as potential treatment targets of the keloids, and finding out the hormone receptors serving as the treatment targets causing the skin keloids through pathological staining and animal experiment verification. According to the method, by revealing the pathological mechanism of keloid formation, the diagnostic marker is identified, and the hormone receptor causing the skinkeloid is found out and serves as a treatment target.

The invention discloses application of bitter gourd exosomes in radiation heart injury protection, the bitter gourd exosomes and rat myocardial cells (H9C2) are co-cultured and subjected to radiation treatment, MTT experiments show that 5-25 [mu] g/ml of the bitter gourd exosomes can promote the cell viability of the radiated H9C2 myocardial cells, and 10 [mu] g/ml of the bitter gourd exosomes are selected as the use concentration of the bitter gourd exosomes. Under the condition that the bitter gourd exosome is 10 [mu] g/ml, Ki-67 fluorescent staining proves that the bitter gourd exosome can promote proliferation of radiated H9C2 myocardial cells. An Annexin V-PE/7-AAD flow apoptosis experiment and an apoptosis protein cleaved-caspase3 immunoblotting test prove that the bitter gourd exosome can be used for reducing the apoptosis of H9C2 myocardial cells after radiation. Immunofluorescence staining and protein immunoblotting of a DNA damage marker gamma-H2A. X show that the bitter gourd exosome can reduce DNA damage of H9C2 myocardial cells after radiation.

The invention discloses a tissue and organ three-dimensional imaging and analysis method based on continuous slicing, multicolor fluorescence and three-dimensional reconstruction. The method mainly comprises the following steps: strictly and continuously slicing a reconstructed object; alternately carrying out multi-color immunofluorescence staining in multiple combinations according to a standardprocess; carrying out multi-channel fluorescence scanning on all the immunofluorescence staining sections to obtain digital section images; aligning continuous slices according to fluorescence channel signals, and inserting various dyeing combinations for three-dimensional reconstruction to obtain a tissue and organ fluorescence three-dimensional model with cell-level resolution; and conducting STL modeling on the interested local area, and conducting various kinds of analysis. According to the method, a high-resolution tissue and organ fluorescence three-dimensional model capable of performing quantitative analysis can be constructed, and the tissue and organ three-dimensional form can be faithfully restored to the maximum extent; one or more fluorescence channels can be checked independently or in an overlapping manner; and STL modeling can be performed on the region of interest to complete spatial quantification and correlation analysis of various cells and functional markers.

The invention belongs to the field of biomedical science, and particularly relates to application of sevoflurane to preparing nerve regeneration promoting medicine. The application has the advantages that neuron regeneration promoted by means of sevoflurane pretreatment is studied and observed by the aid of immunofluorescence staining and laser confocal methods via focal cerebral ischemia models of rats, nerve functional reconstruction procedures after focal cerebral ischemia of the rats are accelerated, as shown by results, large quantities of newborn neurons are available in infarction regions of the rats at early injury stages after the rats are pretreated by the sevoflurane, and the quantities of the newborn neurons are obviously higher than those of untreated groups; synaptic connection and newborn myelin sheath formation are available among newborn neurons of the rats at middle-late injury stages; as proved by the results, effects of promoting post-stroke nerve regeneration and accelerating nerve functional reconstruction of the infarction regions can be realized under pretreatment actions of the sevoflurane; theoretical bases are provided for treating acute nerve injury and neurodegenerative diseases by the sevoflurane; the sevoflurane can be used for preparing the nerve regeneration promoting medicine and is particularly applicable to preparing medicine for promoting nerve regeneration after focal cerebral ischemia injury.

The invention relates to the field of cells, in particular to a preparation method of retinal ganglion cells (RGCs). According to the method, related methods for separating and purifying RGCs are improved, and multiple experiments prove that the number of RGCs obtained from an average single retinal tissue is obviously higher than that of RGCs obtained by an original two-step immune dissociation method, and the purity of the separated RGCs is also obviously higher than that of RGCs obtained by the original two-step immune dissociation method. Specific labeled antibodies beta-tubulin III and BRN3A are used for carrying out immunofluorescence staining on different batches of RGCs. Results show that the purity of the RGCs labeled by two antibodies has no statistical difference, and it is further verified that the improved two-step immune dissociation method can obtain the primary RGCs with relatively stable purity and yield. The research lays a cytological foundation for the large-scale deep exploration of the mechanism of visual impairment caused by RGCs necrosis and apoptosis.

The invention relates to the biotechnical field, and discloses a method for constructing a genetic character chimeric multi-cellar structure. The method comprises the following steps: carrying out immunofluorescence staining on cell communities with different genetic backgrounds and needing chimericity, and observing to determine whether the above cells have intercellular junctions or not; mixing the cells with the intercellular junctions, adding a culture solution, placing the obtained product in a low protein adsorption centrifuge tube, allowing the centrifuge tube to stand at room temperature for 10-20min in order to make the cells precipitate to the bottom of the centrifuge tube, taking out the cells and the culture solution, placing the obtained product in a container, and growing to form a three dimensional structure with functions; and respectively labeling the cells with different genetic backgrounds, having no intercellular junctions and needing junctions by complementary nucleic acid single strands connected with NHS, mixing the labeled cells, placing the obtained product in the container, and growing to form the three-dimensional structure with functions. The method has the advantages of operation time shortening, simplicity, efficiency improvement and good chimeric effect.

The invention belongs to the technical field of biology, and particularly relates to application of a novel kidney-warming sperm-producing drink for promoting early embryonic development by maintaining the low level of H3K27me3. The method comprises the following steps: molding sexually mature male mice with cyclophosphamide, drenching the male mice with a new kidney-warming sperm-producing drink for 30 days, mating the treated male mice with female mice subjected to superovulation treatment, and counting the developmental rate of embryos in each stage; detecting the expression of H3K27me3 in sperms, prokaryotic embryos and 2-cell embryos by using an immunofluorescence staining method; detecting the expression of histone demethylase KDM6A and methylated transferase EZH2 in a 2-cell embryo with development retardation by using western blotting; using the qRT-PCR for analyzing the expression of zygote genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2 and MYC) in the 2-cell embryo with development retardation. According to the analysis of the results, the new application of the new kidney-warming sperm-producing drink for promoting early embryonic development by maintaining the low H3K27me3 modification level in sperms and male pronucleus is found.