IF Troubleshooting

Potential cause	Recommended test
Conditions of antibody are not optimized.	Titrate the antibody concentration to optimize best working conditions. Incubate the primary antibody at room temperature or at 4°C overnight.
Protein of interest is low expressed in used cells.	Use signal amplification when visualizing
Damaged epitope.	Over-fixation, reduce fixative step, or change to another fixative.
Antibody is not suitable for detection of protein in its native form in IF.	Perform a test on a native Western Blot (not-denaturated).

Potential cause	Recommended test
Target of interest is a nuclear protein.	Use a permeabilization step.
Weak or no fluorescent signal.	Store fluorescent-labeled antibody in the dark. Use a direct-labeled primary antibody. Use signal enhancer
Fading signal	Store fluorescent-labeled antibody in the dark. Choose another mounting media
Artifacts	Can be due to: cell culture, cell density, insufficient washing, over-fixation, mounting issues.

Potential cause	Recommended test
Non-specific binding of primary/ secondary antibodies.	Run control. Prolong blocking step
The sample is poorly washed.	Repeat or prolong washing step.
The antibody incubation temperature is too high.	Incubate at 4°C.
Inappropriate fixation causes artifacts or damages the antigen.	Reduce fixative step. Change fixative.
Permeabilization has damaged the cell or protein.	Decrease or skip the permeabilization step.
The slide has dried.	Always keep slide moist.