A method for immunofluorescence staining of neurospheres

Abstract

The invention discloses a method for immunofluorescence staining of neurospheres: (1) neurosphere cleaning: take suspension cultured neurospheres, centrifuge, discard the supernatant, resuspend the neurospheres, centrifuge again, discard the supernatant; (2) fix ; (3) permeabilization and blocking; (4) primary antibody incubation; (5) rinsing; (6) secondary antibody incubation; On the glass slide, anti-fluorescence quenching agent was added dropwise, covered with a cover glass, and the neurospheres were flattened and mounted for fluorescence microscope observation. The present invention utilizes the characteristics of large diameter of neurospheres and is visible to the naked eye, directly adopts low-speed centrifugation to collect cells at the bottom of the tube, avoids the loss of neurospheres during the dyeing process, can maintain the original cell shape and internal structure of neurospheres, and effectively avoids the process of cell climbing The changes in the antigen characteristics caused by the process; the physical extrusion method is used to directly compress the neurospheres into thin films, without slicing, and has low requirements for equipment.

Description

A method for immunofluorescence staining of neurospheres technical field [0001] The present invention relates to a method for immunofluorescence staining of neurospheres, belonging to the technical field of molecular biology. Background technique Neural stem cells refer to the ability to differentiate into neurons, astrocytes and oligodendrocytes. I renew and can provide a large number of cell populations of brain tissue cells. Neural stem cells are mostly obtained by the method of suspension neurosphere culture. Get and research. Immunofluorescence technology is to label the fluorochrome that does not affect the activity of the antigen-antibody on the antibody (or antigen), After the corresponding antigen (or antibody) is combined, a specific fluorescent reaction is presented under a fluorescence microscope, thereby carrying out tissue or Intracellular localization of antigenic substances. [0004] Immunofluorescence staining of neurospheres is usually first perf...

AI Technical Summary

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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