| Problem |
Cause |
Recommendation |
Background is too high |
The quality of the primary antibody is not high |
Use highly specific and high-titer primary antibodies |
Background is too high
|
The concentration of the primary/secondary antibody is too high |
Reduce primary/secondary antibody concentrations |
Background is too high
|
The sealing is not complete |
Increase the duration of protein blocking |
Background is too high
|
The washing is not sufficient |
Increase the number and duration of washes |
Background is too high
|
The background can be reduced by adjusting the parameters of the fluorescence microscope. |
|
Weak or no signal |
No target protein or very low expression |
Use cells or tissues with high expression levels |
Weak or no signal
|
Poor cell permeability |
Increase the duration or concentration of the permeabilization agent |
Weak or no signal
|
Primary/secondary antibody concentration too low |
Increase its concentration |
Weak or no signal
|
Wrong secondary antibody selection (species mismatch) |
Use a secondary antibody that is matched to the species of the primary antibody |
Fluorescent light that goes out quickly |
Fluorescein itself has poor light stability due to its intrinsic properties |
Use a fluorescent secondary antibody with good light stability |
Fluorescent light that goes out quickly
|
Seal with a common sealing agent |
Seal the film with a fluorescent quencher |
Cells have spontaneous fluorescence |
Glutaraldehyde is used for fixation |
Fluorescence quenching is performed after fixation and before fluorescent staining, such as with NaIO4, NaBH4, glycine, etc., and the level of autofluorescence is checked before staining |
Cells have spontaneous fluorescence
|
The material itself (e.g., paraffin) has autofluorescence |
Set up a negative control to eliminate background staining by reducing the parameters of the fluorescence microscope |
Cells have spontaneous fluorescence
|
The content of molecules (e.g., riboflavin, cytochrome) in the cell components that can produce autofluorescence is high; the ratio of dead cells/live cells in the cultured cells is high |
|
