Floating sphere immunofluorescent staining method and staining device

Abstract

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.

Description

technical field [0001] The invention belongs to a method for coloring a test sample, in particular to an immunofluorescence staining method and a staining device for suspended cell spheres. Background technique [0002] Immunofluorescent staining is a commonly used experimental method in cell and molecular biology. It applies the principle of antigen-antibody reaction and uses fluorescent substances to label antibodies for antigen localization. It is used to observe the expression of target proteins and whether there is co-localization or co-expression relationship between proteins. . [0003] For the common immunofluorescent staining of adherent cells, the commonly used and commonly used method is to "climb" the cells on the surface of the poly-lysine-coated carrier, and then perform the classic staining operation. However, for the suspension cell spheroid commonly used in stem cell research, because it does not adhere to the wall, it is very easy to cause the loss of the ...

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Problems solved by technology

[0005] Chinese patent document CN102288471B discloses a method for immunofluorescent staining of suspension cells. This method is suitable for suspension cells. At least 10 times of cell centrifugation are required to complete the experiment. This method not only consumes time and manpower, but also applies this method to suspension cells. During cell spheroids, due to the different phenotypes of the inner layer cells and the outer layer cells of the cell spheroids, multiple centrifugations can detach the outer layer cells, damage the structure and cell composition of the cell spheroids, and cause the composition of the final test cell spheroids to be different from that before the experimental operation , affecting the accuracy of the experimental results

[0007] Geraldo, S. et al found that immobilizing cell spheres in collagen gel can indeed ensure that the cell spheres will not be lost [8] , but this method still has defects: first, the collagen fibers in the gel will act as a shield, affecting the excitation light intensity and penetration distance of the confocal microscope; second, in order to achieve the same staining effect, it is necessary to The concentration of antibody working solution is doubled or even tripled, and the incubation time of antibody is prolonged, which increases the time consumed and experimental funds [9]

[0008] Guerrero-Cazares, H. et al. studied cell spheroids embedded with OCT embedding agent to make frozen tissue sections and then performed immunofluorescence staining [10] , the disadvantage of this method is that a cryostat is required, and the slices of frozen tissues need to be prepared by professional technical personnel, and it is difficult to find the layer with cell spheres in the process of slicing, and the experimental steps are cumbersome and time-consuming.

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