Immunocytochemistry/ Immunofluorescence (ICC/IF)
Load positive and negative controls to ensure the ICC/IF procedure is performed correctly. ICC/IF results can vary depending on the protocol, so we recommend following the provided procedure fir.
- Weak or no signal
Possible reasons:
1) Target protein is not expressed, or is expressed at low levels, in cells or tissue
Consult the literature to confirm cell/tissue expression of the target protein and perform a western blot to detect the target protein in the target cell or tissue. As an alternative, use a transfected cell line.
2) Poor detection of intracellular protein due to insufficient cell permeabilization
Increase incubation time in permeabilization buffer. Use Triton X-100 or NP40 to permeabilize cell if the target protein is located in the nucleus.
3) Epitope compromised by improper fixation method prior to staining
Choose the best method for sample fixation. For example, 4% paraformaldehyde may be more appropriate for membrane-associated proteins. Organic solvents (e.g., methanol or ethanol) may not be suitable for membrane-associated proteins.
4) Insufficient concentrations of primary or secondary antibody
Adjust the concentration of primary antibody or secondary antibody. Confirm that the primary and secondary antibodies are compatible (i.e., if your primary antibody was raised in rabbit, use a secondary antibody that reacts with rabbit).
5) Insufficient incubation time with the primary antibody
Adjust the incubation time with the primary antibody (e.g., incubate overnight at 4°C).
6) Inactive fluorophore on secondary antibody
Store fluorophore-conjugated antibodies in the dark. Always protect the secondary antibody from light during the experiment.
7) Excessive washing
Wash the slides gently and do not soak in washing buffer for extended periods of time.
8) Excessive blocking
Ensure that the appropriate blocking buffer is being used.
9) Excessive salt concentration in the binding or washing buffers
Reduce the salt concentration in the binding or washing buffers.
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- High background and/or non-specific staining
Possible reasons:
1) Overexpression of the target protein in cells or tissues
Please note that overexpression of the protein may lead to aberrant localization..
2) Concentration of the primary or secondary antibody is too high
Reduce the concentration of the primary or secondary antibody.
3) Non-specific binding of secondary antibody
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
Incubate the sample with primary antibody at 4°C.
5) Excessive incubation time
Decrease the time of incubation with primary or secondary antibody.
6) Over-fixation
Reduce the time of fixation.
7) Improper blocking reagent used
Select a suitable blocking reagent. Make sure that the IgG in blocking reagent (i.e., in normal serum or in some BSA preps) will not cross-react with the secondary antibody.
8) Insufficient washing
Increase the number and/or duration of washing steps.
9) Tissue sections/cells have dried out
Maintain the samples in humid conditions during the experiment.
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