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FAQs & solutions for fluorescent antibody staining
Unable to label cells 1. Please check if the antibodiesare properly preserved in correct condition (Store at 4°C and protect from exposure to light);
2. Please check if the antibodiesare expired;
3. Please check if the correct antibody(primary antibody or secondary antibody) is added into samples;
4. Please ensure the antibody has bound fluorescent substance. If the antibody doesn’t bind fluorescent substance, please ensure to use appropriate fluorescently labeled secondary antibody;
5. Please check if the secondary antibody is still active---Has it ever been used with other primary antibody together?
6. Please check ifthe secondary antibody can recognize the primary antibody?
7. For PE or APC labeled antibody, please ensure the samples shouldn’t store under frozen condition;
8. Please check if the samples can express the target antigen. It is advised to establish a group of samples expressing the antigen as positive control;
9. Please ensure if the species of sample is matched with that of antibody;
10. Please ensure if the correct length of exciting light and correct analysis channel are used.
Lowfluorescence intensity 1. Please check if the antibody is diluted excessively. It is suggested to dilute antibody properly according to the handbook of antibody;
2.The possible reason for weakly positive during indirect staining is prozone phenomenon, that is a false negative response resulting from high antibody titer which interferes with regular formation of antigen- antibody lattice, it may bring about weak staining result. Solution: It is suggested to dilute the antibody;
3. For this problem, too many cells also bring about weak positive result. It is suggested to adjust the concentration of cells according to the handbook;
4. It is probably because of the expression level of antigen. Please find out the expression level of antigen according to previous research articles. If the antigen is weakly expressed, please try to use the fluorescently labeled antibody with strong fluorescence;
5. Antibody with cross reaction is easier to bring about weakly positive result, comparing to specific antibody;
6. Please optimizethe incubation time and incubation temperature of primary and secondary antibodies.
Non-specific staining 1. For non-specific staining, itmay be caused by cell auto-fluorescence. Solution: Under same detecting condition, it needs to deploy blank control in order to ascertain the level of cell autofluorescence.
2. It is suggested to choose CD16/32 antibody (Product ID: M10161) to enclose Fc receptor, so that it can reduce non-specific binding of antibody and cell.
3. For non-specific staining, the usage of secondary antibody may bring about such problem. It is suggested to select secondary antibody which has no cross reaction with detected tissue.
4. Please ensure if the washing after staining is sufficient.
5. It is suggested to reduce the concentration of antibody, for the sake of reducing non-specific staining
PE labeled antibody brings no staining result, but using FITC to label the same antibody shows satisfying result of staining.
1. Please ensure if the PE labeled antibody is stored in low temperature. If it were not stored in low temperature, it is recommended to buy new antibody.
2. Paraformaldehyde fixation may bring about the problem. It is suggested to prepare the paraformaldehyde fixation solution just when you need it; Alternatively, after the treatment of cells, analyze the cells as soon as possible without the need for fixation.
Scattering signal is abnormal
1. Please try to use fresh cells. Scattering signal is visible in the dead cells