Immunocytochemistry/ Immunofluorescence
Please load a positive and negative control to ensure the IF procedure is performed correctly. The result of immunofluorescence can vary with experimental procedure, so we recommend that users perform experiments using the protocol we provide.
- High background and/or non-specific staining
There are several possible reasons:
1) Overexpression of the target protein in cells or tissue
Please note that the overexpression of protein may lead to aberrant localization.
2) The concentration of primary or secondary antibody is too high
Please reduce the concentration of primary or secondary antibody.
3) Non-specific binding of secondary antibody
Please include a “secondary antibody only” control, in which slides are incubated in the presence of secondary antibody alone (without primary antibody).
4) High temperature during incubation with primary antibody
Please incubate the sample with primary antibody in 4°C.
5) Excessive incubation time
Please reduce the time of incubation with primary or secondary antibody.
6) Over-fixation
Please reduce the time of fixation.
7) Improper blocking reagent used
Please choose the suitable blocking reagent. Make sure that the IgG in blocking reagent (e.g., BSA or normal serum, etc.) will not cross react with secondary antibody.
8) Insufficient washing
Please increase the number and/or duration of washing.
9) The sections/cells have dried out during experiment
Please make sure the sections/cells are always in humid condition during experiment.
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- Weak or no signal
There are several possible reasons:
1) The target protein is not expressed or expressed at low levels in cells or tissue.
Please review the reference and run the western blot for the protein of interest to check its expression in target cell or tissue. As an alternative, use a transfected cell line.
2) Poor detection of intracellular protein due to insufficient cell permeabilization
Please increase the incubation time in permeabilization buffer. Use Triton X-100 or NP40 to permeabilize cell if the target proteins are located in the nucleus.
3) Eipitope destroyed by Improper fixation method prior to stain
Please choose the suitable method for sample fixation. For example, 4% paraformaldehyde may be more suitable for membrane-associated proteins. Organic solvents (e.g., methanol or ethanol, etc.) may not be not suitable for membrane associated proteins.
4) Insufficient concentrations of primary or secondary antibody used
Please increase the concentration of primary antibody or secondary antibody. Make sure the primary and secondary antibodies are compatible (e.g., if your primary antibody is made in rabbit, use a secondary antibody that reacts with rabbit).
5) Insufficient incubation time with the primary antibody
Please increase the incubation time with the primary antibody (e.g., Incubate overnight at 4°C).
6) Inactive fluorophore of secondary antibody
Please store fluorophore-conjugated antibodies in the dark. Always protect the secondary antibody from light during experiment.
7) Excessive washing
Please wash the slides gently and do not soak in washing buffer for extended periods of time.
8) Excessive blocking
Please ensure that the appropriate blocking buffer is being used.
9) Excessive salt concentration in the binding or washing buffers
Please reduce the salt concentration in the binding or washing buffers.
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