No or Weak Staining
| Reason | Solution |
| Low expression level of the protein | For target proteins with low expression levels, it is advisable to use fluorescent dyes with high intensity in staining. Alternatively, indirect staining using primary and secondary antibodies can be attempted to enhance detection sensitivity. |
| Unclear expression of the target protein in the sample | Positive controls with clearly expressed target proteins can be used, while cells lacking expression of the target protein or knockout (KO) cells can serve as negative controls. |
| Lack of blocking agents during detection of secreted factors | For secreted proteins such as cytokines and chemokines, blocking agents such as monensin, brefeldin A, or a combination of both can be used to retain the factors inside the cells. |
| Improper choice of fixation and permeabilization reagents | When staining cytoplasmic and nuclear targets, appropriate fixation and permeabilization reagents should be used to ensure better detection of cytoplasmic and nuclear proteins |
| Improper storage of antibodies | Strictly follow the recommended conditions for antibody storage as stated in the instructions. |
| Quenching of fluorescent dyes | Fluorescent-labeled antibodies and samples stained with fluorescent-labeled antibodies should be stored protected from light. |
| Improper antibody concentration | Optimal antibody concentration for the experiment can be determined through antibody titration. |
| Inadequate incubation time and temperature | Time and temperature for antibody incubation can be referred to the recommended conditions in the instructions, and appropriate gradient adjustments can be made based on the experiment. |
| Mismatch between primary and secondary antibodies | For indirect immunofluorescence staining, it is important to ensure proper matching between the fluorescent secondary antibody and the primary antibody. |
| Incorrect choice of filters | Check the excitation and emission wavelengths of the fluorescent dyes used to ensure the correct laser and filters are used for data acquisition. |
| Excessive compensation of data | Set compensation using single-stained controls and fluorescence minus one (FMO) controls for each experiment. |
| Incorrect gating of cell populations | Ensure proper gating of cell populations. |
High Background or Nonspecific Staining
| Reason | Solution |
| High autofluorescence | Cellular autofluorescence depends on cell type and may also be influenced by cell preparation methods. Use samples stained under the same stimulation conditions but without any reagents as controls for cellular autofluorescence. |
| Presence of dead cells | When performing surface staining on live cells, use live cell viability dyes (such as PI or 7-AAD) to gate out dead cells. However, when staining fixed cells, use fixable live cell viability dyes that can withstand fixation and be used for intracellular staining. |
| Insufficient Fc receptor (FcR) blocking | Use Fc receptor (FcR) blocking reagents or Fab fragments to mitigate the effects of FcR. |
| Excessive antibody concentration | Use the recommended antibody dosage as stated in the instructions, and titrate the antibody concentration based on the experimental conditions. |
| Prolonged antibody incubation time | The antibody incubation time can be referred to the manufacturer's recommended conditions, and appropriate gradient adjustments can be made based on the experiment. |
| Insufficient washing | Increase the times of wash after staining. |
| Inadequate compensation adjustment | Set compensation using single-stained controls and FMO controls for each experiment. |
Other Abnormal Staining
| Problem | Reason | Solution |
| False Positives | Presence of cell aggregates or dead cells in the analysis | Use single-cell gating and employ viability dyes to exclude cell aggregates and dead cells. |
| Poor specificity of the antibody | Replace the antibody with a more specific one. | |
| Signal from isotype control antibody is higher than the detection antibody | Incorrect concentration of the isotype control antibody | Ensure that the concentration of the isotype control antibody matches the detection antibody. |
| Isotype control antibody is from a different manufacturer | Use an isotype control antibody from the same manufacturer as the detection antibody. |
