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Immunofluorescence (IF) Troubleshooting Guide

Weak Or No Signal

Possible Cause


Inappropriate storage of samples; signal may fade if fluorophores are exposed to light for extended periods of time

Perform incubations and store samples in the dark. Samples may be mounted in an anti-fade solution, such as ProLong® Gold Antifade Reagent #9071. Samples should be imaged immediately following mounting for the best results.

Cell/tissue samples stored for too long

Use freshly prepared slides/plates to avoid loss of antigenicity.

Inadequate fixation

Consult the CST product datasheet for the recommended protocol; remove media and quickly/thoroughly wash in fixative immediately after treatment. For phospho-specific antibodies, use at least 4% formaldehyde to inhibit endogenous phosphatases.

Incorrect antibody dilution (antibody too dilute)

Consult the CST product datasheet or for the recommended antibody dilution.

Not using the recommended incubation time

Primary antibody incubation according to a rigorously tested protocol provides consistent, reliable results. CST antibodies have been developed and validated for optimal results when incubated at 4°C overnight.

Inappropriate testing model

If possible, protein expression should be confirmed by western blot or other means.

Target protein not induced properly

Optimal treatment conditions and controls should be determined for each target.

Low expression of protein of interest

Modify detection approach; consider signal amplification or pair with a brighter fluorophore.

Wrong permeabilization method

Consult datasheet for recommended protocol.

Incorrect use of secondary antibody

Use recommended concentration and check secondary antibody is matched to host species of the primary antibody.

Wrong excitation wavelength

Ensure illumination and detection (laser/excitation/emission filter) matches excitation wavelength of fluorophore(s).

Low signal in multiplexed IHC

Optimization of deparaffination, antigen retrieval, and signal amplification methods.

High Background

Possible Cause


Sample autofluorescence

Use unstained samples as controls to check autofluorescence levels. Check datasheet for correct fixation reagent. Old fixative may autofluoresce; replace old formaldehyde stocks and prepare fresh dilutions. Use EM-grade glutaraldehyde freshly diluted from ampules. Choose longer wavelength channels for low-abundance targets.

Insufficient blocking

Use normal serum from the same species as the secondary antibody used. Consider a charge-based blocker, such as Image-iT® FX Signal Enhancer #11932, depending on the source of background.

Incorrect antibody dilution (primary or secondary antibody too concentrated)

Consult the CST product datasheet or for the recommended antibody dilution.

Samples dried out

It is vital that sample remains covered in liquid throughout the staining procedure.

Insufficient washing

Wash to remove excess fixative, excess secondary antibody, and loosely bound, non-specific antibody interactions.

Secondary cross-reactivity

Use isotype control antibodies to determine whether your secondary antibody is cross-reacting.

Non-specific antibody binding

If available, compare to knockdown (siRNA) or knockout cells, or compare to cells known to express higher or lower levels of the target antigen.