Immunoglobulin staining method for neurosphere

Abstract

The invention discloses an immunoglobulin staining method for a neurosphere. The method comprises the steps of (1) neurosphere cleaning: taking neurospheres in suspension culture, centrifuging, discarding supernatant, resuspending the neurospheres, centrifuging again, and discarding supernatant, (2) fixing, (3) permeability and blocking, (4) primary antibody incubation, (5) rinsing, (6) secondaryantibody incubation, (7) rinsing, and (8) mounting: adding a PBS buffer solution, resuspending the neurospheres, dropwise dropping the neurospheres on an object slide, dropwise adding an anti-fluorescence quencher, covering with cover glass, flattening and packaging the neurospheres, and observing with a fluorescence microscopy. According to the method, cells are collected at a tube bottom by directly using low-speed centrifugation by using the characteristics of large diameter and visibility for a naked eye of the neurospheres, the loss of the neurospheres in a staining process is avoided, the original cell morphology and internal structure of the neurospheres can be maintained, the change of antigenic properties caused in a cell climbing process is effectively avoided, the neurospheres are directly pressed by using a physical pressing method, the neurospheres are pressed into a film shape, the cutting is not needed, and the requirements for equipment are low.

Description

technical field [0001] The invention relates to a neurosphere immunofluorescent staining method, which belongs to the technical field of molecular biology. Background technique [0002] Neural stem cells refer to a cell group that has the ability to differentiate into neurons, astrocytes and oligodendrocytes, can self-renew and can provide a large number of brain tissue cells. Neural stem cells are mostly obtained and studied by the method of suspension neurosphere culture. [0003] Immunofluorescence technology is to label the antibody (or antigen) with a fluorescent dye that does not affect the activity of the antigen and antibody, and after binding to the corresponding antigen (or antibody), a specific fluorescent reaction is presented under a fluorescent microscope, thereby performing tissue or cell localization of antigenic substances. [0004] Immunofluorescent staining of neurospheres is usually carried out on cell slides or frozen sections of neurospheres, so that ...

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Problems solved by technology

[0004] Immunofluorescent staining of neurospheres is usually carried out on cell slides or frozen sections of neurospheres, so that the neurospheres are fixed on the glass carrier, and then stained. The steps are complicated, the cost is high, and the professional requirements for personnel are high.

In addition, in the process of cell climbing, there are disadvantages such as difficulty in cell adhesion, easy to change antigen characteristics and interfere with experimental results; frozen section production has disadvantages such as high equipment requirements, easy rolling and stripping, etc.

[0005] Chinese invention patent CN 102288471 A discloses a method for immunofluorescence staining of suspension cells. This method is suitable for suspension cells, but the immunofluorescence staining of neurospheres is insufficient. The reasons are: first, the diameter of neurospheres is relatively large. When staining, only the surface cells of the neurospheres can be stained, and the cells inside the neurospheres cannot be successfully stained; secondly, this method is easy to cause cell lysis, and for neurospheres, the original cell morphology and internal structure will be destroyed, thereby affecting the staining results. The application value is not high

Chinese invention patent CN 105424450 A discloses a suspension cell spheroid immunofluorescence staining method and a staining device. This method is suitable for suspension cell spheroids. This method requires a special staining device for cell spheroid immunofluorescence staining, which is difficult for general laboratories to meet. ; This method is permeabilized and blocked separately, which is time-consuming; the blocking solution of this method is goat serum, and the blocking effect is poor, which is easy to cause non-specific

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