1. Ensure that all the primary and secondary have been correctly stored and have activity;
2. Set a positive control of known antigen expression to expel other unexpected reasong that may result in false-negative;
3. Don't freeze your materials if the fluorescent dye is phycoerythrin or allophycocyanin-based.
1. Maybe the concentration of antibody is too low. Ensure you have used the suitable concentration;
2. Maybe the number of cells is too high. Adjust cell density to recommended one;
3. Maybe the incubated time and temperature with antibody is not suitable, which should be optimizaed;
4. Maybe the antigen expression is weak. Select an antibody that is conjugated to a brighter fluorochrome, such as PE and PerCP.
1. Maybe the concentration of antibody is too low, which should be optimized carefully;
2. In indirect IF staining, select a secondary antibody that do not have cross-react with your material;
3. Make sure that you have blocking nonspecific binding sites and have adequate washing steps.
1. Make sure that your reagents used don't affect the activity or structure of your antigens;
2. If your tested antigens are expressed intracellularly, some cell permeabilization methods should be used.