Immunofluorescence Staining: A Typical Workflow


Immunocytochemistry is a very sensitive method that requires a lot of experience and optimization. Slight changes in the protocol can markedly alter the results. If you get a low signal, no signal, or a high background, please consult the following troubleshooting guide.

High Background

Reason Solution
Inappropriate or too long fixation, leading to artefacts Reduce fixation time or change the fixative
Insufficient blocking Prolong the incubation time, consider using a different blocking solution (we recommend using serum from the secondary antibody host)
No specificity of the primary antibody Use a primary antibody that is proven to work for immunofluorescence in the chosen model system; if available, use a knockdown/knockout sample as a negative control
Too high primary/secondary antibody concentration, too long incubation time, too high incubation temperature Optimize the antibody concentration and incubation time/temperature, consult manufacturer’s protocol
Cross-reactivity of the secondary antibody Use isotype controls for the secondary antibody to check for cross-reactivity
Not enough washing Verify that all washing steps are carried out properly; if necessary, prolong the washing steps
Bleaching during imaging Use a secondary antibody conjugated to a fluorophore suitable for your chosen microscopy technique
Low signal intensity, resulting in noise Optimize the signal-to-noise ratio, e.g., by using a brighter fluorophore for detection; if applicable, increase the expression of the antigen of interest (e.g., by overexpression or by addition of inducing agents)
High autofluorescence Check the sample autofluorescence by using unstained controls; use fresh fixation solutions (expired formalin solutions might have a high autofluorescence); use materials with low autofluorescence (e.g., ibidi µ-Slides or Chamber Slides); use mounting medium with low autofluorescence (e.g., ibidi Mounting Medium / ibidi Mounting Medium With DAPI)

Low Signal or Lack of Signal

Reason Solution
Drying out of the sample Always keep the sample moist
Overfixation of the sample, leading to epitope damage Reduce fixation time or change the fixative
Inadequate permeabilization method Optimize or skip the permeabilization step
No binding of primary antibody to the antigen of interest Use a primary antibody that is proven to work for immunofluorescence in the chosen model system; check the antibody functionality by using a positive control (e.g., by overexpression or by addition of inducing agents)
Too high primary/secondary antibody dilution, too short incubation time, too low incubation temperature Increase the antibody concentration or the incubation time/temperature; consult manufacturer’s protocol
Very low or no antigen expression Use a positive control (e.g., an overexpression model); reconsider your experimental system
Inappropriate microscopy detection method Use a more sensitive method for image acquisition; check your used filter/laser setup; use a brighter fluorophore for detection

Read on and learn more about a typical Workflow of an Immunofluorescence Staining, or about how to do Immunofluorescence Stainings With the ibidi Chambers.